CN109439598A - One streptomycete and its application - Google Patents
One streptomycete and its application Download PDFInfo
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Abstract
The present invention relates to a streptomycete and its applications.The bacterial strain is thunder lattice streptomycete Streptomyces regensis, and deposit number is CGMCC No.16746.It can be used for field of biological control, figured cloth moths attracted by lamplight, fall webworms, diamondback moth, Ostrinia furnacalis, Tetranychus urticae, cabbage caterpillar, lopper worm, potato bug, black peach aphid and Dolycoris baccarum prevented and treated.The streptomycete has many advantages, such as that insecticidal activity is high, insecticidal spectrum is wide, genetic stability is good, and bacterial strain fermentation liquor has that strong to temperature tolerance, insecticidal activity is stable, good light stability, shelf-stable, has good development and application values.
Description
Technical field
The invention belongs to technical field of biological control, are related to a streptomycete and its application.
Background technique
Actinomyces are the quasi-microorganisms that earliest discovery has Biocontrol Effect, report a variety of chains in actinomyces in succession both at home and abroad
Mould can generate new pest-resistant antibiotic, and the discovery of avermectin and successfully exploitation are considered as that antibiotic is answered in agricultural production
Third milestone is antibiotic most potential in agricultural production.In more than the 3000 kinds of antibiotics reported in recent years,
Antiinsect antibiotic accounts for about 5%.Have found some antibiotic kinds with development potentiality.But existing antibiotic is generally existing to kill
Worm low efficiency, the problems such as stability is poor.Therefore, a kind of efficient, stable novel streptomycete or its biological and ecological methods to prevent plant disease, pests, and erosion product pair are developed
Agriculture and forestry sustainable development is most important.
Summary of the invention
In view of the problems of prior art, the present invention provides a kind of novel streptomycete, which has desinsection
The features such as active high, insecticidal spectrum is wide, genetic stability is good；Bacterial strain fermentation liquor have to temperature tolerance it is strong, insecticidal activity is stable,
The advantages that good light stability, easy storage, there are fine development and application values.
The technical scheme to solve the above technical problems is that
The present invention provides a streptomycete, which is thunder lattice streptomycete Streptomyces regensis, and preservation is compiled
Number be CGMCC No.16746.
In this application, inventor is named as LKY208.The bacterial strain is preserved in China on November 15th, 2018
Microbiological Culture Collection administration committee common micro-organisms center (CGMCC), the collection address are Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1.
Bacterial strain riotous growth on Gause I culture medium, aerial hyphae is luxuriant, when 28 DEG C of 1~2d of culture, bacterium colony light
It is sliding, generated without spore, 3d starts visible gas silk yellow green, pistac, faint yellow, and matrix color gradually becomes yellow after 4d
Color.It observes under the microscope, substrate mycelium is not broken without horizontal film；Electronic Speculum is observation indicate that fibrillae of spores is short, often irregularly,
Accidental short and loose-screw shape, spore ellipse, cylindricality, surface is smooth or irregular fold.
Inventor is exploring streptomycete in the breakthrough of field of biological control (such as biological control of figured cloth moths attracted by lamplight), from adopting
Isolated 217 plants of actinomyces carry out screening and control efficiency measurement from the soil of Jilin Province different regions, are finally obtained 1
Strain can effectively prevent the antagonistic strain of figured cloth moths attracted by lamplight larva, be named as LKY208, and to its fermentation liquid stability and bacterium
Strain classification position has carried out system research, and new bio-control factors are provided for biological control.Inventor has found under study for action, the strepto-
Bacterium has the characteristics that insecticidal activity is high, insecticidal spectrum is wide, genetic stability is good；Bacterial strain fermentation liquor have to temperature tolerance it is strong, kill
The advantages that worm is activity stabilized, good light stability, shelf-stable has fine development and application values.
The present invention also provides application of the above-mentioned streptomycete in agriculture and forestry injurious insect prevention and treatment.The pest can be selected from figured cloth lamp
Moth, fall webworms, diamondback moth, Ostrinia furnacalis, Tetranychus urticae, cabbage caterpillar, lopper worm, potato bug and black peach aphid, spot palpus
The combination of one or more of stinkbug.
Streptomycete provided by the invention has many advantages, such as that insecticidal activity is high, insecticidal spectrum is wide and genetic stability is good.By reality
Verifying, for above-mentioned figured cloth moths attracted by lamplight, fall webworms, diamondback moth, Ostrinia furnacalis, Tetranychus urticae, cabbage caterpillar, lopper worm,
Potato bug, black peach aphid all have insecticidal action.It is wherein apparent to the pest insecticidal effect of Lepidoptera.To fall webworms larva
Have a higher insecticidal activity, 48h corrected mortality is 72.1%, to the corrected mortality point of cabbage caterpillar and lopper worm 48h
Not Wei 43.9% and 36.5%, also there is preferable insecticidal activity to Tetranychus urticae, the corrected mortality of 48h is up to 56%；To small
The insecticidal activity of diamond-back moth larva is only second to figured cloth moths attracted by lamplight larva and fall webworms larva, corrected mortality 69.5%；In particular, right
The insecticidal activity highest of Lepidoptera figured cloth moths attracted by lamplight larva, the corrected mortality of 48h is up to 74.1%.
The present invention also provides a kind of biological prevention and control agents, the fermentation liquid including above-mentioned streptomycete or above-mentioned streptomycete.The bacterium
Strain has the characteristics that insecticidal activity is high, insecticidal spectrum is wide, genetic stability is good；Bacterial strain fermentation liquor have to temperature tolerance it is strong, kill
The advantages that worm is activity stabilized, good light stability, shelf-stable has fine development and application values.
The present invention also provides a kind of application of above-mentioned biological prevention and control agent in control of insect.The pest be selected from figured cloth moths attracted by lamplight,
In fall webworms, diamondback moth, Ostrinia furnacalis, Tetranychus urticae, cabbage caterpillar, lopper worm, potato bug and black peach aphid, Dolycoris baccarum
One or more of combinations.
The present invention provides a kind of preparation method of above-mentioned biological prevention and control agent, comprising the following steps: above-mentioned streptomycete of fermenting is obtained
Obtain fermentation liquid.
Specifically, it may comprise steps of: above-mentioned streptomycete being seeded on Gause I slant medium and is cultivated,
Pure culture biscuits involvng inoculation to fermentation medium is cultivated again, collects fermentation liquid later.
In practical application, (such as freeze-drying) fermentation liquid can also be prepared by the way of dry for convenience of storing
Powder before to be used, is applied on object to be prevented and treated again after powder is dissolved with solvent.
Further, the cultivation temperature is 25 DEG C~28 DEG C.The temperature is conducive to the growth of above-mentioned streptomycete.
The present invention can provide the application method of above-mentioned biological prevention and control agent, which comprises the following steps: by above-mentioned life
Anti- preparation is applied on the plant for needing to prevent and treat.
Detailed description of the invention
Fig. 1 is the result of influence of the temperature to bacterial strain fermentation liquor insecticidal activity.
Fig. 2 is the result of influence of the pH to bacterial strain fermentation liquor insecticidal activity.
Fig. 3 is the result of influence of the illumination to bacterial strain fermentation liquor insecticidal activity.
Fig. 4 is the result of the influence of storage time and temperature to bacterial strain fermentation liquor insecticidal activity.
The result of influence of Fig. 5 squamous subculture to bacterial strain fermentation liquor insecticidal activity.
The electromicroscopic photograph of Fig. 6 bacterial strain LKY208.
The phylogenetic tree of Fig. 7 bacterial strain LKY208 and related strain.
Specific embodiment
The principle and features of the present invention will be described below with reference to the accompanying drawings, and the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the invention.
For the present invention by artemia primary dcreening operation and figured cloth moths attracted by lamplight larva secondary screening, 1 plant is obtained from soil has higher insecticidal activity
Actinomycetes strain, named LKY208, for the biocontrol effect and classification position of the clearly bacterial strain, using blade infusion process,
Feed contamination method, slide infusion process and conventional dip method determine it to figured cloth moths attracted by lamplight, fall webworms, diamondback moth, Asia respectively
Corn borer, Tetranychus urticae, cabbage caterpillar, lopper worm, potato bug, black peach aphid, 10 kinds of agriculture and forestry injurious insects of Dolycoris baccarum insecticidal activity；
Primary Study has been carried out to the stability of fermentation liquid simultaneously, and its classification position is had studied by 16SrRNA.The result shows that: it should
Bacterial strain is in addition to Dolycoris baccarum, and 9 kinds of selected insect sources such as figured cloth moths attracted by lamplight, fall webworms, diamondback moth have lethal effect, and insecticidal spectrum is wide,
Wherein most strong to the lethal effect of figured cloth moths attracted by lamplight, the corrected mortality of 48h is up to 70% or more, and bacterial strain fermentation liquor is to temperature tolerance
By force, stablize in 28~50 DEG C of insecticidal activities；Degrees measurement shows that bacterial strain insecticidal activity is preferable in the range of pH4~7, ferments
The photostability of liquid, depot and bacterial strain genetic stability show stabilization, have preferable development and application values；Through 16SrRNA
Sequence alignment analysis identifies that bacterial strain LKY208 is thunder lattice streptomycete Streptomyces regensis, is now preserved in the micro- life of China
Object culture presevation administration committee common micro-organisms center (CGMCC), deposit number are CGMCC No.16746, this is domestic
Report the prevention and treatment that streptomycete is applied to the pests such as figured cloth moths attracted by lamplight for the first time outside.
It is introduced below by specific embodiment.
1 materials and methods
1.1 material to be tested
1.1.1 for trying soil
Since in June, 2016,30 parts of soil sample are acquired in the different regions in Jilin Province.See Table 1 for details
Table 1
1.1.2 strains tested streptomycete bacterial strain LKY-208 is isolated from the soil of number 15, by this laboratory screening, separation
And save, detailed process sees below embodiment.
1.1.3 selected insect source
Figured cloth moths attracted by lamplight (Camptoloma interiorata, Walker), Lepidoptera, Arctiidae；
Fall webworms (Hyphantria cunea, Drury), Lepidoptera, Arctiidae；
Diamondback moth (Plutella xylostella L.), Lepidoptera, plutellidae；
Ostrinia furnacalis (Ostrinia furnacalis Guenee), Lepidoptera, Pyralidae；
Tetranychus urticae (Tetranych usurticae Koch), Acarina, Tetranychidae；
Cabbage caterpillar (Pieris rapae L.), Lepidoptera, Sulfur butterfly；
Lopper worm (Mamestra brassicae L.), Lepidoptera, Noctuidae；
Potato bug (Epilachna vigintioctomaculata Motschulsky), coleoptera, ladybirds；
Black peach aphid (Myzus persicae Sulzer), Homoptera, Aphidiadae；
Dolycoris baccarum (Dolycoris baccarum L.), Homoptera, Pentatomiddae；
The above selected insect source be the generation phase acquired in woodland and greenhouse and insectary in raise.
1.1.4 for trying artemia (BrineShrimp)
Artemia (Brine Shrimp), also known as salt water fairy shrimp are purchased from Youfa Aquatic Product Co. Ltd., Binzhou Harbour, Shandong Prov..
1.1.5 for trying culture medium
Gause I synthetic media, formula: soluble starch 20g, K2HPO40.5g, KNO31g, MgSO4·7H2O
0.5g, NaCl 0.5g, FeSO4·7H2O 0.01g, agar 20g, distilled water 1000mL, pH7.0~7.2.
Liquid fermentation medium, formula: groundnut meal 2.5%, soluble starch 5.0%, yeast powder 0.08%, glucose
0.02%, (NH4)2SO40.08%, NaCl 0.2%, CaCO30.32%, pH 7.0~7.2.
Artificial seawater, the bibliography (separation screening and ocean desinsection chain of Xiong Lixia marine microorganism insecticide active substance
The research Chinese environmental ecological Studies institute doctoral thesis of mould L173,2004.) it prepares, formula are as follows: NaCl 24.4770g,
MgCl2·6H2O 4.9810g, Na2SO43.9170g CaCl2·H2O 2.230g, NaHCO30.1920g, KCl
0.6640g, NaBO30.0260g, SrCl20.0240g, NaF 0.0039g, 1000mL.
Embodiment 1
1.2.1 collecting soil sample
After choosing appropriate place, surface soil is removed with scuppit, takes soil about 30~50g of the depths 5~10cm, is packed into sterilizing
Paper bag in, number, and indicate collecting location and acquisition the date, the separation for streptomycete.
1.2.2 the isolation and purification of streptomycete
Using plating dilutions partition method, (bibliography: Fang Zhongda plants sick Beijing research method [M]: Chinese agriculture is published
Society, 2007,243-250) carry out streptomycete separation and purifying.For the pollution for reducing miscellaneous bacteria, especially bacterium, will first weigh up
10g soil toasts 1h at 120 DEG C, is then added in 100mL sterile water, after shaking 30min, draws 1mL suspension, use is sterile
Water gradient dilution, from 10-3、10-4With 10-50.1mL is drawn in dilution tube respectively to be added on Gause I plate, even spread, so
Culture dish is inverted in 28 DEG C of insulating boxs 3~10d of culture afterwards, daily observation and the different bacterial strain of choosing colony form and
When be transferred on Gause I inclined-plane and cultivate, after purifying 3~5 times again using isolation by dilution method, number and save to 4 DEG C of refrigerators
In it is spare.
1.2.3 shake flask fermentation primary dcreening operation insecticidal activity bacterial strain
The preparation of fermentation liquid: the streptomycete isolated and purified in 1.2.2 is forwarded on Gause I slant medium, and 28 DEG C
After lower culture 7d, beaten in pure culture biscuits involvng inoculation to the 250mL triangular flask that 40mL Medium of shaking flask fermentation is housed using 7mm punch, 28
DEG C, 150r/min, shaken cultivation 5d, 4000r/min are centrifuged 20min, take supernatant to cross biofilter removal thallus spare.
The measurement of insecticidal activity: artemia liquid assay method is used.When measurement, artemia eggs is placed in artificial seawater (pH8~
9) it, activates for 24 hours for 28 DEG C.It is separately added into 0.1mL artemia liquid (about 20~30 artemias) in 96 porocyte culture plates and 0.1mL is waited for
The fermentation liquid of bacterial strain is surveyed, after 28 DEG C of cultures for 24 hours, sem observation is dissected with binocular and counts the artemia death rate, separately set Gause I liquid
Body culture medium is control.
1.2.4 shake flask fermentation secondary screening insecticidal activity bacterial strain
Selected insect source: figured cloth moths attracted by lamplight pick up from Daliang City Jin Hushan, this laboratory continuously rearing.
Insecticidal activity assay: using blade infusion process (bibliography: the first from left ring, Wang Kaiyun, Jiang Xingyin .4 kind antibiotics
Virulence and ovicidal action [J] pesticide of the insecticide to diamondback moth different larval instar larva, 2004,43 (1): 26-27).Take fresh illiteracy
Ancient oak blade is soaked with bacterial strain fermentation liquor, is placed in the culture dish of sterilizing, and 2 age figured cloth moths attracted by lamplight larvas, every ware 20 are accessed after drying
Head, 3 repetitions set the not inoculum fermented liquid culture medium of dipping separately as blank control (CK).Be placed on artificial climate incubator (25 ±
1 DEG C, RH 70%~80%, photoperiod 14h/d) in raising, record dead borer population amount after 48h, calculate separately the death rate and correction is dead
Die rate.
Embodiment 2
1.2.5 the measurement of streptomycete LKY208 fermentation liquid broad-spectrum insecticidal activity
To the insecticidal activity assay of figured cloth moths attracted by lamplight, fall webworms, diamondback moth, cabbage caterpillar and lopper worm: being soaked using blade
Stain method (the same 1.2.4 of method).
To the insecticidal activity assay of corn borer: using feed contamination method (bibliography: Xu Shulan, Li Hui, Chen Qijin sweet tea
Influence [J] the Changjiang river vegetables of the Pathogenic Tests and group rearing of dish californica nuclear polyhedrosis virus to viral yield, 2010 (18):
10-12.).The feed (an about daily diet for larva) for uniformly cutting a certain size, after bacterial strain fermentation liquor is admixed high pressure sterilization
Man-made feeds in (100 μ L/g), feed 2 age corn borer larvaes, 20 larvas of every processing are repeated 3 times.Rearing conditions are same as above,
Test worm existence or death condition are checked after 48h under binocular anatomical lens, the death rate is calculated and corrected mortality, man-made feeds is mixed clearly
Water is control.
To the insecticidal activity assay of Tetranychus urticae: using slide infusion process (bibliography: FAO.Revised Method
for Spider Mites and Their Egg(e.g.Tetranychus spp.and Panonychus ulmi Koch)
[J].FAO Plant Production and Protection,1980(21):49-54.).Double faced adhesive tape paper is cut into 2~
The length of 3cm is attached to one end of clean microscopic slide, the scraps of paper on viscose glue is thrown off with tweezers.With writing brush gently picking
Female adult mite in the same size, body colour is fresh, action is active, its back is adhered on double faced adhesive tape.It is careful not to glue when paste
The firmly foot and mouthpart of mite, so that its limbs can activity.Every slide glues Tetranychus urticae 25, is repeated 4 times.Tetranychus urticae will be stained with
Glass slide be placed on 25 ± 1 DEG C, RH be 70~80% artificial climate incubator in place 4h after, with binocular vision and pick
Except dead or torpescence individual.Then the glass slide one end for being stained with Tetranychus urticae is immersed in fermentation liquid, is taken after gently shaking 5s
Out, mite body and liquid extra around are blotted with filter paper, then is placed in above-mentioned artificial climate incubator and raises.After 48h, double
Test worm existence or death condition are checked under mesh anatomical lens, calculate the death rate and corrected mortality.Clear water is separately impregnated with viscous mite slide
For control.
To the insecticidal activity assay of black peach aphid: using blade infusion process.From field, acquisition has the cabbage leaves of test worm, with zero
Number writing brush removes sundries.Aphid and host's blade are put into bacterial strain fermentation liquor together and impregnate 5s, and is gently shaken.Blade takes out
Afterwards, surplus liquid is sucked with filter paper, after drying at room temperature, is put into the culture dish that diameter is 9cm, is placed in artificial climate incubator
Middle raising.30 aphids of every processing, are repeated 3 times, and are control with clear water dipping.Check that test worm is raw after 48h under binocular anatomical lens
It deposits or death condition, calculates the death rate and corrected mortality.
The insecticidal activity assay of potato bug and Dolycoris baccarum: using conventional dip method (bibliography: the ocean Liu Jining
The insecticidal activity of 050101 bacterial strain of microorganism is screened and its Tropical Agricultural University Of South China master thesis is studied in genetic recombination,
2004.).It will be put into for examination insect in leaching worm device, 30s is soaked in bacterial strain fermentation liquor, is put into foster worm bottle after taking-up, cultural method
Ibid.It every processing adult 20, is repeated 3 times, is control with test worm clear water dipping, rearing conditions are same as above, and are dissected after 48h in binocular
Test worm existence or death condition are checked under mirror, the death rate and corrected mortality are calculated,.
Embodiment 3
1.2.6 thermal stability determination
Equivalent bacterial strain fermentation liquor is taken to handle respectively at 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C and 100 DEG C
It is taken out after 1h, after natural cooling, insecticidal activity detection is carried out to 2-3 age figured cloth moths attracted by lamplight larva using leaf dipping method, with untreated
(25 DEG C) of fermentation liquid are control.
Embodiment 4
1.2.7pH Stability Determination
Take equivalent bacterial strain fermentation liquor with 1mol/LHCL and 1mol/LNaOH adjust separately pH be 1,2,3,4,5,6,8,9,
10,11,12,13 and 14, standing recalls to original pH7 afterwards for 24 hours, and it is living to carry out desinsection to 2-3 age figured cloth moths attracted by lamplight larva using leaf dipping method
Property detection.It is control with untreated fermentation liquid.
Embodiment 5
1.2.8 light durability measures
Equivalent bacterial strain fermentation liquor is taken to be respectively placed under daylight after irradiation 1h, 2h, 3h, 4h, 5h, using leaf dipping method to 2-3 age
Figured cloth moths attracted by lamplight larva carries out insecticidal activity detection, is control with untreated fermentation liquid.
Embodiment 6
1.2.9 storage property measures
It is after taking equivalent bacterial strain fermentation liquor to store 15d under 4 DEG C, 40 DEG C, -20 DEG C of constant temperature respectively, fermentation liquid is extensive
Again to original volume, insecticidal activity detection is carried out to 2-3 age figured cloth moths attracted by lamplight larva using leaf dipping method, with untreated fermentation liquid (25
DEG C) it is control.
Embodiment 7
1.2.10 bacterial strain genetic stability measures
Streptomycete LKY208 is forwarded on Gause I slant medium, F is denoted as1In generation, cultivates 5~6d under the conditions of 28 DEG C
Afterwards, then inclined-plane of transferring, it is denoted as F2In generation, successively subculture switching is until F6Generation.Take the above-mentioned F for cultivating 7d respectively1、F2、F3、F4、F5、F6
For inclined-plane culture body, ferment by above-mentioned fermentation process.Each generation bacterial strain fermentation liquor is detected to 2-3 age figured cloth using leaf dipping method
The insecticidal activity of moths attracted by lamplight larva.
Embodiment 8
1.2.11 bacterial strain is identified
Strain is inoculated on No. 1 agar plate of Zhi Gaoshi, in the medium with 45o oblique cutting by sterile cover slip, 28 DEG C of trainings
After supporting 5~7d, coverslip is taken out, is directly observed under an optical microscope；Meanwhile selecting mycelia growth density moderate, but gas is raw
Mycelia and the well-developed part of fibrillae of spores, after vacuum evaporating film-making, with scanning electron microscopic observation spore chain and spore shape and table
Face structure, the method in processing method bibliography.(bibliography are as follows: Yang Rui, Wang Qi, Zhang Lu wait actinomyces scanning electron microscope
Sample preparation methods comparative studies [J] electron microscopic journal, 2014.33 (1): 84-89.)
Reference resin typeTMGenome DNA extracting reagent kit (purchases in Tiangeng biochemical technology Co., Ltd) specification extraction
DNA, to extract DNA as template, design primer expands 16S rDNA, upstream primer sequence are as follows: 5'-
AGAGTTTGATCCTGGCTCAG-3'(SEQ ID NO.1), downstream primer sequence are as follows: 5'-GGTTACCTTGTTACGACTT-3'
(SEQ ID NO.2) is synthesized by precious biological (Dalian) Engineering Co., Ltd.50 μ L PCR reaction systems: 25 μ of PCR Mixture
L, each 1 μ L of 2 μ L of Template, upper and lower primer, ddH2O 21μL.PCR reaction condition: 94 DEG C of denaturation 5min；94 DEG C of denaturation
1min, 56 DEG C of renaturation 1min, 72 DEG C of extension 2min, 35 circulations；72 DEG C of extension 5min, 4 DEG C of preservations are produced with the PCR of sterile water
Object is negative control, and using the bacterial strain PCR product of known 16S rDNA as positive control, it is solidifying to carry out 2% agarose by 2 μ L of applied sample amount
Gel electrophoresis.Recovery product send biological (Dalian) Engineering Co., Ltd of most valuable treasure to carry out bidirectional sequencing, and sequence is through BioEdit 7.0.1 etc.
After software analysis and manual correction, the sequence measured downloaded in GenBank with the blast program of NCBI known to it is similar
The sequence of type culture carries out tetraploid rice analysis, and with the adjacent method of 6.0 software of MEGA (neighbor-joining,
NJ it) carries out sequence alignment and drawing system develops tree graph.
Above-described embodiment carries out data statistics using SPSS17.0, and DuncanShi duncan's new multiple range method carries out significance of difference inspection
It tests.
The result and analysis of 2 the various embodiments described above
The isolation and purification of 2.1 streptomycetes
Streptomycete being widely distributed in the soil, in conventional separation process, the quantity of bacterial strain is very big, can not be whole
Retain.Therefore, main to choose colonial morphology and the different bacterial strain tube of pigmentary colours saves in choosing colony, from acquisition
It isolates to obtain 217 streptomyces strains in 30 parts of pedotheques.After purification by 2~3 plates, form color one is obtained
The purifying bacterial strain of cause.These bacterial strains are cultivated in Gause I slant medium, 4 DEG C of preservations are spare as original strain.
2.2 shake flask fermentation primary dcreening operation results
217 isolated streptomyces strains are subjected to shake flask fermentation.By artemia determination of activity, 11 plants are obtained to halogen
Worm lethality is up to 50% or more bacterial strain, wherein number LKY208 bacterial strain, effect is best, and up to 94.7% (table 2), primary dcreening operation is obtained
The active bacterial strain tube obtained, separately saves backup.
The primary dcreening operation result of 2 insecticidal activity streptomycete bacterial strain of table
Data are average ± standard deviation in table 2.
2.3 shake flask fermentation secondary screening results
There is the streptomycete bacterial strain of greater activity to carry out shake flask fermentation culture artemia for what primary dcreening operation obtained, prepares its fermentation
Liquid carries out insecticidal activity assay by target of figured cloth moths attracted by lamplight larva, the observation after 48h the results show that bacterial strain LKY208 have compared with
High insecticidal activity is significantly higher than other strains testeds.Above-mentioned bacterial strains fermentation liquid to the corrected mortality of figured cloth moths attracted by lamplight larva all
In 70% or more (table 3).
The secondary screening result of 3 insecticidal activity streptomycete bacterial strain of table
Data are average ± standard deviation in table 3.It indicates with lowercases different after column data through the new multipole of DuncanShi
Difference method inspection is significant in the level difference of P < 0.01, similarly hereinafter.
The measurement of 2.4 streptomycete LKY208 fermentation liquid insecticidal activities
Bacterial strain LKY208 fermentation liquid is determined respectively to the insecticidal activity of different agriculture and forestry injurious insects.Result of study shows bacterial strain
LKY208 fermentation liquid is to the insecticidal activity highest of Lepidoptera figured cloth moths attracted by lamplight larva, and the corrected mortality of 48h is up to 74.1%, to beauty
The corrected mortality of the white moth of state is only second to figured cloth moths attracted by lamplight larva and fall webworms up to 72.1%, to the insecticidal activity of diamondback moth larvae
Larva, corrected mortality 69.5%, compared with other test worms, significant difference also has preferable desinsection living to Tetranychus urticae
Property, the corrected mortality of 48h is up to 56%；Corrected mortality to Lepidoptera cabbage caterpillar and lopper worm 48h is respectively 43.9%
With 36.5%；But the desinsections such as homoptera pest black peach aphid, Hemipteran pest Dolycoris baccarum and coleopteran pest potato bug are made
It is obvious (as shown in table 4) to the insecticidal action of other pests with being not so good as.
The measurement result of 4 bacterial strain LKY208 fermentation liquid insecticidal activity of table
2.5 streptomycete LKY208 fermentation liquids and strain stability measurement
2.5.1 thermal stability determination
After treatment of different temperature, the insecticidal activity of bacterial strain LKY208 fermentation liquid is slightly changed.When temperature is lower than 50 DEG C, hair
Zymotic fluid is preferable to the insecticidal activity of figured cloth moths attracted by lamplight, maintains good stability, but when temperature is higher than 50 DEG C, insecticidal effect has
Decline (Fig. 1), even if temperature reaches 100 DEG C, LKY208 fermentation liquid still has insecticidal activity.
2.5.2pH Stability Determination
In 1~14 range of pH, desinsection work of the fermentation liquid of bacterial strain LKY208 with the change of pH to figured cloth moths attracted by lamplight larva
Property varies slightly.The insecticidal activity highest of fermentation liquid when pH is 7, when pH is less than 7, fermentation liquid insecticidal activity is slightly reduced,
But when pH is more than 9, the insecticidal activity of bacterial strain fermentation liquor significantly reduces (as shown in Figure 2), thus bacterial strain fermentation liquor meta-acid with
It is activity stabilized under neutrallty condition, and it is apparently higher than fermentation liquid under alkaline condition.
2.5.3 light durability measures
The fermentation liquid of bacterial strain LKY208 is after the processing of different light application times, to the insecticidal activity the change of divergence of figured cloth moths attracted by lamplight
It is smaller (as shown in Figure 3), illustrate that the insecticide active substance in bacterial strain LKY208 fermentation liquid is more stable in illumination condition, is not easy point
Solution.
2.5.4 storage property measures
After the fermentation liquid of bacterial strain LKY208 saves 15d 50 DEG C, 4 DEG C, -20 DEG C, 25, to the insecticidal activities of figured cloth moths attracted by lamplight with
Control is compared, and activity is almost without reduction.Therefore bacterial strain fermentation liquor has storing properties, can be store with long-term preservation with 4 DEG C of low temperature
Hide effect preferably (as shown in Figure 4).
2.5.5 genetic stability measures
Test result shows insecticidal activity and original bacteria of the fermentation liquid to figured cloth moths attracted by lamplight of streptomycete LKY208 squamous subculture
Strain is compared, and extremely strong genetic stability (as shown in Figure 5) is demonstrated by.
The identification of 2.6 bacterial strains
2.6.1 bacterial strain LKY208 Morphological Identification
Bacterial strain LKY208 riotous growth on Gause I culture medium, aerial hyphae is luxuriant, when 28 DEG C of 1~2d of culture, bacterium
Fall it is smooth, generated without spore, 3d starts visible gas silk yellow green, pistac, faint yellow, and matrix color gradually becomes after 4d
Yellow.It observes under the microscope, substrate mycelium is not broken without horizontal film；Electronic Speculum is not advised often observation indicate that fibrillae of spores is short
Then, accidental short and loose-screw shape, spore ellipse, cylindricality, surface is smooth or irregular fold (as shown in Figure 6), according to shape
State feature preliminary analysis bacterial strain LKY208 is streptomyces.
The 16S rRNA of 2.6 bacterial strain LKY208 is identified
After carrying out PCR amplification, purifying, sequencing to the 16S rDNA gene order of bacterial strain LKY208, its sequence length is measured
For 1388bp.Compared with the 16S rDNA sequence of GenBank database, with thunder lattice streptomycete Streptomyces regensis
Homology highest, similar value is 99% or more.Selection and higher 10 bacterial strains of bacterial strain LKY208 homology, establish system
Development tree, the results showed that, bacterial strain LKY208 and Streptomyces regensis strain NBRC (GenBank:
4 bacterial strains such as NR112402.1), gather in a branch, and affiliation is nearest, therefore Preliminary Identification bacterial strain LKY208 is Lei Gelian
Mould (MH473142) (as shown in Figure 7).
3. conclusion and discussion
With the continuous development that biological control of insect pests is studied, insecticide active substance has been found from the metabolite of microorganism
As one of the hot spot studied both at home and abroad.Actinomyces are widely distributed in nature, and metabolite type is abundant, are to generate antibiosis
The most quasi-microorganism of plain class active material, and streptomycete is that a most category of antibiotic is generated in actinomyces.Therefore,
Efficient insecticide active material producing bacterial strain is screened from streptomycete has extraordinary development and application prospect.
Soil is the important place of streptomycete perch, and it is novel that screening, which obtains and generates the streptomycete of active material, from soil
Essential prelimi nary work in antiinsect antibiotic research and development.And the streptomycete resource of nature is very huge,
Think therefrom to screen ideal biocontrol bacterial strain, workload is also very huge, and result is not expectable.Traditional desinsection antibiosis
The screening of element is all the test insect using targeted insect as screening test, since the action target spot of targeted insect is extensive,
Be conducive to the detection of various active insecticidal components, but relatively labor intensive and material resources.It is carried out using small-sized living animal
Early stage primary dcreening operation, then carrying out secondary screening with model related with structure or mechanism is the screening strategy being widely recognized as at present.
The application is isolated from collected all kinds of pedotheques to 217 streptomycetes, with halogen using isolation by dilution method
Worm is primary dcreening operation instruction biology, by shake flask fermentation primary dcreening operation, obtains 11 plants of streptomycete bacterial strains to artemia lethality up to 50% or more,
Insect is indicated by secondary screening of figured cloth moths attracted by lamplight, has screened the bacterial strain LKY208 with higher insecticidal activity, LKY208 pairs of streptomycete
The insecticidal effect of figured cloth moths attracted by lamplight is obvious, and the corrected mortality of 48h has research and development prospect well up to 70% or more.
Various indoor bioassay methods are utilized simultaneously, determine streptomycete LKY208 bacterial strain fermentation liquor to various insects
Insecticidal effect.The result shows that insecticidal activity highest of the bacterial strain LKY208 fermentation liquid to figured cloth moths attracted by lamplight, the corrected mortality of 48h
Up to 74.1%, there is preferable insecticidal activity to fall webworms, the corrected mortality of 48h is 72.1%；To cabbage caterpillar and sweet
Blue noctuid also has certain bioactivity；But the insecticidal actions such as black peach aphid, Dolycoris baccarum and potato bug are not so good as to other evils
The insecticidal action of worm is obvious.This may be because the insect killing substance studied of this test is generated by microbial metabolism, and it is micro-
For the secondary metabolite of biology to the toxic action of insect selectivity with higher, general one kind secondary metabolite may be only
There is effect to some kinds of insect.
To bacterial strain LKY208 fermentation liquid carry out stability test the result shows that, bacterial strain LKY208 fermentation liquid 50 DEG C with
Under, pH be that insecticidal activity is more stable under slant acidity and neutrallty condition；With good light durability, keeping quality and heredity
Performance is stablized.Therefore, bacterial strain LKY208 has good potential research and development value, and the insect killing substance that fermentation generates is being developed
There is good prospect in terms of biological insecticides.
With the rapid development of molecular genetics and molecular biotechnology, the taxology of actinomyces is from classical form
It has been sorted into the molecular classification epoch.16SrDNA has become a molecular indexes at present, is widely used to various microorganisms
Hereditary feature and molecular difference research, can be by the measurement and comparative analysis of the DNA sequence dna to unknown microorganism, quickly
And effectively achieve the purpose of taxonomic identification.Wherein 16SrDNA is common to biological cell, and function is homologous and most ancient, loses
Pass that relatively stable, representative information amount is moderate, molecular size is suitble to operate, it is to grind that its sequence variation and evolutionary distance, which is adapted,
The good material for studying carefully phyletic evolution, it is more and more important in classification of actinomycetes identification work.
This test using copy number of the 16SrDNA in different streptomycetes, fragment length and base sequence homology pair
Bacterial strain LKY208 is classified identification, and bacterial strain LKY208 is named as streptomyces thunder lattice streptomycete (Streptomyces
Regensis), and it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), is protected
Hiding number is CGMCC No.16746.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Sequence table
<110>Jilin Inst. of Forestry Science
<120>one streptomycetes and its application
<160> 2
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 19
<212> DNA
<213>artificial sequence
<400> 2
ggttaccttg ttacgactt 19
Claims (10)
1. a streptomycete, which is characterized in that the bacterial strain is thunder lattice streptomycete Streptomyces regensis, and preservation is compiled
Number be CGMCC No.16746.
2. application of the streptomycete described in claim 1 in agriculture and forestry injurious insect prevention and treatment.
3. applying according to claim 2, which is characterized in that the pest be selected from figured cloth moths attracted by lamplight, fall webworms, diamondback moth,
The group of one or more of Ostrinia furnacalis, Tetranychus urticae, cabbage caterpillar, lopper worm, potato bug and black peach aphid, Dolycoris baccarum
It closes.
4. a kind of biological prevention and control agent, which is characterized in that including streptomycete described in claim 1 or strepto- described in claim 1
The fermentation liquid of bacterium.
5. application of the biological prevention and control agent described in claim 4 in control of insect.
6. applying according to claim 5, which is characterized in that the pest be selected from figured cloth moths attracted by lamplight, fall webworms, diamondback moth,
The group of one or more of Ostrinia furnacalis, Tetranychus urticae, cabbage caterpillar, lopper worm, potato bug and black peach aphid, Dolycoris baccarum
It closes.
7. a kind of preparation method of biological prevention and control agent described in claim 4, which comprises the following steps: fermentation right is wanted
Streptomycete described in claim 1 is asked, fermentation liquid is obtained.
8. preparation method according to claim 7, which comprises the following steps: claims are required 1 institute
The streptomycete stated is seeded on Gause I slant medium and cultivates, then pure culture biscuits involvng inoculation to fermentation medium is cultivated, it
After collect fermentation liquid.
9. preparation method according to claim 7, which is characterized in that the cultivation temperature is 25~28 DEG C.
10. a kind of application method of biological prevention and control agent described in claim 4, which comprises the following steps: by claim
4 biological prevention and control agents are applied on the plant for needing to prevent and treat.
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