CN110004069A - A kind of Strain of Beauveria bassiana and its application - Google Patents
A kind of Strain of Beauveria bassiana and its application Download PDFInfo
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- CN110004069A CN110004069A CN201910398058.9A CN201910398058A CN110004069A CN 110004069 A CN110004069 A CN 110004069A CN 201910398058 A CN201910398058 A CN 201910398058A CN 110004069 A CN110004069 A CN 110004069A
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Abstract
The invention discloses a kind of Strain of Beauveria bassiana and its application, the Strain of Beauveria bassiana classification naming isBeauveria bassiana, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation day: on December 28th, 2018, deposit number: CGMCC No.17068, the beauveria bassiana belongs to mycota, Fungi Imperfecti door, Hyphomycetes, Moniliales, Moniliaceae, Beauveria.Using the application for the Strain of Beauveria bassiana in preparation biological control trident maple material bark beetle biological agent.
Description
Technical field
The invention belongs to microorganisms to be separately cultured technical field, and in particular to a kind of Strain of Beauveria bassiana and its application.
Background technique
The important Landscape Tree Species trident maple in Kunming is by bark beetle (roundlet chest bark beetleEuwallacea fornicatus)
Harm seriously destroys the construction of Kunming Scenery of gardens and causes huge economic loss, very urgent to the improvement of bark beetle.
It is difficult to find, lives in addition hidden since bark beetle body is small, host provides good barrier for its completion history of life, causes
Prevention and treatment is difficult；And roundlet chest bark beetle is because its host plant is numerous and widely distributed, difficulty of prevention and cure it is bigger (Walgama R S. etc.,
2012).Bark beetle is only active in the external world within a period of time of new adult, and most times seek hidden life in life, leads to
Often mating, feeding under bark.It can be seen that its harm has concealed spy according to the biological characteristics that bark beetle is shown
Point, for eating into the prevention and treatment of the bark beetle in host tree species for a long time, using Agro-chemicals control, often effect is bad, and chemical prevention has
Environmental pollution this big drawback.And the radiation in physical control method and the stifling prevention and treatment for being directed to quarantine bark beetle,
The prevention and treatment of living body host plant it is not suitable for, the physical method control worm effect of sick branch trimming is also not very good yet.And biology is anti-
Method is controlled, is the raising with country to environmental consciousness, becomes the green control method gradually approved by people.Entomogenous fungi is
The important factor of pest population Natural regulation and the important materials of biological control of insect pests, using entomogenous fungi pest control, by
Step become it is a kind of with biological control be dominate comprehensive treatment important channel.It is intended that the biological control of the pest provides strain money
Source, this research by be separately cultured out from dead bark beetle adult can be lethal to bark beetle illness bacterial strain as biocontrol microorganisms.
The strain isolation is tested from dead bark beetle by tieback, through statistical analysis, has remarkable effect to material bark beetle lethal effect
Bacterial strain, be beauveria bassiana through morphology and Molecular IdentificationBeauveria bassianaBBXDCSFJ03, in this, as three
The effective biocontrol bacterial strain that angle maple material bark beetle filters out.
Summary of the invention
The first object of the present invention is to provide a kind of Strain of Beauveria bassiana；Second is designed to provide the ball spore
The application of muscardine bacterial strain.
Outer unless otherwise indicated, percentage employed in the present invention is percentage by volume.
The first object of the present invention is achieved in that the beauveria bassianaBeauveriabassiana, it is named as
BBXDCSFJ03 is identified as beauveria bassianaBeauveriabassiana, it is from the lethal trident maple material bark beetle body of natural occurrence
It is upper isolated, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on December 28th, 2018
(China General Microbiological Culture Collection Center, abbreviation CGMCC.Address are as follows: north
The institute 3 of the Chaoyang District Jing Shi North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode 100080).Its deposit number is
CGMCC No:17068, the beauveria bassiana belong to mycota, Deuteromycotina, Hyphomycetes, Moniliales, Moniliaceae,
Beauveria.
Beauveria bassiana of the present inventionBeauveria bassianaBBXDCSFJ03 is named as BBXDCSFJ03, is
It is isolated from dead trident maple material bark beetle of falling ill naturally, Chinese microorganism strain is preserved on December 28th, 2018
Preservation administration committee General Microbiological Culture collection (China General Microbiological Culture
Collection Center, abbreviation CGMCC.Address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and the Chinese Academy of Sciences is micro-
Biological study institute, postcode 100080).Its deposit number is CGMCC No:17068
The bacterial strain is good to the causative effect of trident maple material bark beetle, can penetrate trident maple material bark beetle (roundlet chest in a short time
Bark beetleEuwallacea fornicatus) at body segment, and parasitic triangular maple material bark beetle and breed.
The bacterial strain can quickly be bred after touching trident maple material bark beetle.
The bacterial strain produces spore in trident maple material bark beetle body surface, produces spore later in trident maple material bark beetle (roundlet chest bark beetleEuwallacea fornicatus) body surface collection conidium, tieback to trident maple material bark beetle.It is verified by Koch's Postulates,
Tieback bacterial strain conidium infects other health experiments and has same virulence lethal effect with trident maple material bark beetle.
The bacterial strain is obtained by following specific steps:
A, trident maple material bark beetle acquires: acquiring back trident maple material bark beetle (the roundlet chest bark beetle that withered trident maple branch strips out healthEuwallacea fornicatus) living worm body and the polypide that dies of illness naturally, it takes back in laboratory and raises.
B, strain isolation and screening: high pressure-temperature sterilizing institute experimental material in need, and operated in aseptic operating platform
All steps.
The ethanol solution that matched proportion density is 75% is cleaned nature to die of illness trident maple material bark beetle 5 seconds, is then 0.1% by proportion
Mercuric chloride solution to polypide surface sterilization 2-3 minutes, the slow shower of sterile water 3 times, with the tweezers of sterilizing by trident maple material bark beetle
Worm (roundlet chest bark beetleEuwallacea fornicatus) body head, chest, abdomen different parts separate, and will separate after three
Maple material bark beetle different physical feeling in angle is carefully placed into culture medium.
Culture medium is common PDA culture medium.
It is transferred in 25 DEG C of ± constant incubators together and cultivates 5-7d.Repeat purifying twice.
C, fungi preservation: trident maple material bark beetle pathogenic strain is carried out in laboratory in time and is saved, in picking propagating culture medium
Vigorous strain inoculated is grown into Storaged media, stationary culture 7 days in 25 DEG C of ± dark, is frozen in 4 DEG C of refrigerators；It saves
Culture medium is PDA culture medium；By strain inoculated to the common test tube slant PDA in aseptic operation box, put after 25 DEG C of ± culture 7d
Into being saved in 4 DEG C of refrigerators.
PDA solid state rheology based component is calculated as with g/L: 160~240g of potato, 10~20g of glucose, 15~20g of agar,
PH is natural.
PDA solid state rheology based component is preferred in terms of g/L are as follows: potato 200g, glucose 20g, agar 20g, PH are natural.
Collection of specimens described in step A is from each Grand Duchy Triangle ID such as Kunming Green Lake Park, Heilongtang park, botanical garden
Maple plantation.
Collection of specimens described in step A is in each Grand Duchy Triangle ID maple kind such as Green Lake Park, Heilongtang park, botanical garden
Plant garden trident maple returns deadwood item.
Collection of specimens described in step A is under drying, the environment of illumination abundance.
Indoor raising described in step A is to return deadwood item with worm.
Step is in aseptic operating platform described in step B, operates under alcolhol burner, and calcination is inoculated on alcolhol burner flame flame envelope
Needle, oese operate all separating steps under waiting around alcohol.
Sterilizing described in step B is to set the test material requested such as transfer needle, oese, sterile water, tweezers, culture medium
In high steam formula sterilizer, sterilization treatment 30 minutes or more at 121 DEG C.
Step is in aseptic operating platform described in step B, is seeded to culture medium, and imported from America sealed membrane seals all breeding trainings
Support base.
Breeding condition of culture is preferred described in step B；Stationary culture 7-9d in 25 DEG C of ± dark.
The condition of preservation culture described in step C is preferred；25 DEG C of ± dark stationary culture 7-9d.
One plant of beauveria bassianaBeauveria bassianaFungal bacterial strain BBXDCSFJ03 and its in biological control triangle
The method of maple material bark beetle, activation and expansion including bacterial strain are numerous, spore suspension production, cause the inspection of trident maple material bark beetle death virulence
Survey, specifically includes the following steps:
A, bacterial strain activation and expansion are numerous: by beauveria bassianaBeauveria bassianaFungal bacterial strain BBXDCSFJ03 is inoculated into work
Change and expand on breeding culture medium, is generated in 25 DEG C of ± lower culture 7-15d to a large amount of conidiums, obtain activation and the numerous production spore bacterium of expansion
Strain.
B, the preparation of spore suspension: by strain inoculated in PDA culture medium, the constant temperature incubation 7- in 25 DEG C of ± incubators
15d。
The conidium in culture dish is rinsed with the aseptic aqueous solution of addition 0.05%Tween-80.By the bacterium in culture dish
Liquid and spore all pour into cup and do not stop to shake 30min, are filtered with lens wiping paper, finally obtain the spore suspension of no count.
With blood counting chamber, all conidium quantity recorded in internal 400 small lattice under the microscope are placed.Finally calculate institute
Total spore quantity of the spore suspension of preparation, dilution are configured to 1.0 × 107A mL−1Spore suspension.
The preparation method can also include the spore suspension for being fabricated to other concentration.
Blood counting chamber brand described in step B is Shanghai refinement, 16 lattice × 25 lattice blood counting chamber.
Tween-80 described in step B does fungus conidium dispersing agent, Tween-80 polyoxyethylene sorbitan list oil
Acid esters.
Biocontrol effect of the invention includes the following aspects.
1, bacterial strain of the present invention is good to the causative effect of trident maple material bark beetle, can penetrate trident maple material in a short time
Bark beetle (roundlet chest bark beetleEuwallacea fornicatus) at body segment, and parasitize in trident maple material bark beetle body and breed.
2, strain growth of the present invention is rapid, and sporulation quantity is big, is conducive to be mass produced and promote and apply.
3, trident maple material bark beetle (roundlet chest bark beetle of the present inventionEuwallacea fornicatus) biocontrol microorganisms derive from
Trident maple material bark beetle (roundlet chest bark beetle under natural environmental conditionEuwallacea fornicatus) natural sense dies of illness and die separation and obtain
, therefore, it is used, can be as the method for trident maple material bark beetle biological control, and operating process is simple, control efficiency
It is good.
Detailed description of the invention
Fig. 1 is beauveria bassiana in embodiment 1Beauveria bassianaBBXDCSFJ03 is in common PDA culture medium
On, cultivate bacterial strain bacterium colony front form when 7d；
Fig. 2 is beauveria bassiana in embodiment 1Beauveria bassianaBBXDCSFJ03 conidium micrograph；
Fig. 3 is beauveria bassiana in embodiment 5Beauveria bassianaBBXDCSFJ03 conidiophore and mitogenetic spore
Son；
Fig. 4 is beauveria bassiana in embodiment 6Beauveria bassianaBBXDCSFJ03 bacterial strain infects triangle in limb
Maple material bark beetle, the morphological feature of the microscopically observation of growth and development with trident maple material bark beetle；
Fig. 5 is beauveria bassiana in embodiment 6Beauveria bassianaBBXDCSFJ03 infects trident maple material in limb
Bark beetle causes material bark beetle dead.
Specific embodiment
Below with reference to embodiment, the present invention is further illustrated, but the present invention is limited in any way,
Based on present invention teach that it is made it is any transform or replace, all belong to the scope of protection of the present invention.
Strain of Beauveria bassiana classification naming of the present invention isBeauveria bassiana, depositary institution: China
Microbiological Culture Collection administration committee common micro-organisms center (CGMCC), preservation day: on December 28th, 2018, deposit number:
CGMCC No.17068, the beauveria bassiana belong to mycota, Deuteromycotina, Hyphomycetes, Moniliales, Moniliaceae,
Beauveria.
The application of Strain of Beauveria bassiana of the present invention is the Strain of Beauveria bassiana in preparation biological control
Application in trident maple material bark beetle biological agent.
The Strain of Beauveria bassiana preparation biological control trident maple material bark beetle biological agent includes bacterial strain activation and expands
Numerous, spore suspension preparation and biocontrol effect determination step, specifically include:
A, bacterial strain activation and expansion are numerous: the Strain of Beauveria bassiana being inoculated into activation and is expanded on breeding culture medium, in 20 ~ 30 DEG C
Lower culture obtains activation for 10 ~ 20 days and expands numerous production spore bacterial strain；
B, the preparation of spore suspension: by activation and expand in numerous production spore strain inoculated to PDA culture medium, the constant temperature at 20 ~ 30 DEG C
Culture 10 ~ 20 days rinses the spore in culture dish with the aseptic aqueous solution of addition 0.05%Tween-80, by the bacterium in culture dish
Liquid and spore all pour into cup and do not stop to shake 30min, are filtered with lens wiping paper, finally obtain the spore suspension of no count,
It places under the microscope, blood counting chamber counts, and records all spore counts in internal 400 small lattice, finally calculates made
Spore suspension total spore quantity, dilution be configured to various concentration gradient spore suspension；
C, biocontrol effect measures: the filter paper on culture dish middle berth, a little feed is put into each ware, by test worm trident maple material bark beetle
(roundlet chest bark beetleEuwallacea fornicatus) (3, every ware) is put in ware, it is raised at room temperature, it will be real with toothpick
It tests bacterium to choose with polypide, then the feed of picking part bacterium and feeding mixes, so as to wedging together when worm feeding, carry out indoor
Observation；In addition, each processing is repeated 3 times not choose the test worm into bacterium as control (CK), 1-7 days after processing, timing is seen daily
It examines and records polypide morbidity and death condition.
Case is embodied, the present invention will be further described below:
Embodiment 1
Beauveria bassianaBeauveria bassianaAcquisition, identification and the preservation of fungal bacterial strain BBXDCSFJ03.
(1) beauveria bassianaBeauveria bassianaAcquisition, the identification of fungal bacterial strain BBXDCSFJ03.
Beauveria bassiana of the present inventionBeauveria bassianaFungal bacterial strain BBXDCSFJ03 fungi, from triangle
Maple material bark beetle is fallen ill and is obtained on polypide, and trident maple material bark beetle polypide of falling ill picks up from Kunming Green Lake Park, Heilongtang park, plant
Each Grand Duchy Triangle ID maple plantation such as garden, acquisition have back the trident maple branch of withered symptom to take back laboratory dissection, healthy room
Interior raising, the carry out pathogenicbacteria separation culture died of illness.
The ethanol solution that matched proportion density is 75% is cleaned red fire ant 5 seconds, then will match the mercuric chloride solution for 0.1% to worm
Body surface sterilization 2-3 minutes, the slow shower of sterile water 3 times, with the tweezers of sterilizing by trident maple material bark beetle polypide head, chest, abdomen
Different parts separate, and the different physical feeling of the material bark beetle after separating carefully is placed into culture medium and carries out separation training
It supports.
25 DEG C of ± dark stationary culture 7-9d are seeded in propagating culture medium, until bacterium colony is grown.Propagating culture medium is PDA training
Support base.After bacterium colony is grown, the good bacterial strain of picking growing way saves culture into Storaged media, and Storaged media is PDA culture
Base.
PDA solid state rheology based component is preferred in terms of g/L are as follows: potato 200g, glucose 20g, agar 20g, PH are natural.
Further Morphological Identification is carried out by Observation of biological characteristics to above-mentioned isolated bacterial strain PLXDCSFJ04
And Molecular Identification, experimental result record are as follows.
A, morphological feature: beauveria bassiana mycelia has diaphragm, and clear, colorless, bacterium colony is flat, powdery or such as new flour lip pencil,
Superficial white is colourless in potato agar culture basal part to light lacteous.Point stalk of conidiophore or sprig can multiple right angles
Bifurcated, aggregation is agglomerating, and conidiophore and major branch or side shoot inserted part are mostly at right angle.Conidium is spherical, raw producing spore
In zigzag structure made of cell extends, conidiogenous cell is ampuliform changeableization, is tapered upwards by abdomen end.
B, cultural characteristic: in PDA culture medium, in 25 DEG C of ± culture 7-10d colony diameter 5cm, slow growth, powder is purple
Color, reverse side are faint yellow.Initial stage white colony in PDA culture medium, after gradually become pink colour, mycelium edge is in lint shape, mycelia
It is relatively thin.Sporophore is upright, and the bottle of dense arrangement obstructs at branched.Stigma ampuliform is slightly expanded, colyliform branch.By base portion to top
It gradually comes to a point to form very thin neck.Conidium is concatenated, ellipse.Conidium is monospore chain.
C, the stability of bacterial strain: for the bacterium growth temperature range at 15~35 DEG C, suitable growth temperature is 20 DEG C~30 DEG C, raw
For long pH value 5.5~7.5, optimum pH value is 6.5~7.
D, determined dna sequence, the total DNA of the bacterial strain PLXDCSFJ04 5.8S r DNA sequence analysis: are carried out to the bacterial strain
For template, in primer I TS1(5'-TCCGTAGGTGAACCTGCGG3') and ITS4(5'-TCCGTAGGTGAACCTGCGG 3')
Guidance under the PCR amplification bacterial strain 5.8S r DNA sequence dna, PCR reaction system are as follows: 2 × Taq Master Mix, 25 μ L,
Deionized water 20 μ L, primer I TS1 1.5 μ L, primer I TS4 1.5 μ L, DNA 2 μ L, total 50ul.PCR reaction condition: a, 94 DEG C
3 min；B, 94 DEG C of 30 s, 56 DEG C of 40s, 72 DEG C of 50s, 35 circulations；C, 72 DEG C of 10 min, 4 DEG C of preservations.
DNA sequence analysis: PCR reaction product is sent to biotech firm, ITS sequence is sequenced to obtain, through rechecking, in American National
It is compared in Biotechnology Information center (NCBI), comparison result shows bacterial strain BBXDCSFJ03 and beauveria bassianaBeauveria bassianaThe similitude of 5.8S r DNA sequence dna reached 99%.By sequence alignment and morphological feature by bacterial strain
BBXDCSFJ03 is accredited as beauveria bassianaBeauveria bassianaFungal bacterial strain.Bacterial strain of the present invention
Its 5.8S r DNA sequence dna of BBXDCSFJ03 is as follows:
(2) beauveria bassianaBeauveria bassianaThe preservation of fungal bacterial strain BBXDCSFJ03.
By above-mentioned qualification result, confirmation bacterial strain BBXDCSFJ03 is beauveria bassianaBeauveria bassianaFungi
One kind of bacterial strain, number BBXDCSFJ03 are preserved in Chinese microorganism strain preservation conservator on December 28th, 2018
It can General Microbiological Culture collection (China General Microbiological Culture Collection
Center, abbreviation CGMCC.Address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica,
Postcode 100080).Its deposit number is CGMCC No:17068.
Embodiment 2
Beauveria bassianaBeauveria bassianaFungal bacterial strain BBXDCSFJ03 prepares biocontrol agent
(1) preparation of spore suspension.
Firstly, by beauveria bassianaBeauveria bassianaFungal bacterial strain BBXDCSFJ03 is inoculated in common PDA training
It supports on base, the constant temperature incubation 7-15d in 25 DEG C of ± incubators.
PDA solid state rheology based component is preferred in terms of g/L are as follows: potato 200g, glucose 20g, agar 20g, PH are natural.
Then the sterile water of sterilizing is added into 0.05%Tween-80.
Aseptic aqueous solution rinses the spore in culture dish.
By in culture dish bacterium solution and spore all pour into cup and do not stop to shake 30min, filtered with lens wiping paper, it is final
To the spore suspension of no count.
Blood counting chamber is taken out, all spore counts recorded in internal 400 small lattice under the microscope are placed.
Total spore quantity of made spore suspension is finally calculated, dilution is configured to 1.0 × 106A mL−1Spore
Sub- suspension.
(2) result, which is checked and approved, examines.
The spore suspension and the spore suspension after dilution that undiluted concentration is added dropwise in glass slide.It observes under the microscope
Spore quantity on the spore suspension of undiluted concentration and the spore suspension glass slide after dilution, it is obvious poor to detect whether
It is different.
Embodiment 3
Beauveria bassianaBeauveria bassianaFungal bacterial strain BBXDCSFJ03 prepares biocontrol agent.
(1) preparation of spore suspension.
Firstly, by beauveria bassianaBeauveria bassianaFungal bacterial strain BBXDCSFJ03 is inoculated in common PDA training
It supports on base, the constant temperature incubation 7-15d in 26 DEG C of incubators.
PDA solid state rheology based component is preferred in terms of g/L are as follows: potato 200g, glucose 20g, agar 20g, PH are natural.
Then the sterile water of sterilizing is added into 0.05%Tween-80.
Aseptic aqueous solution rinses the spore in culture dish.
By in culture dish bacterium solution and spore all pour into cup and do not stop to shake 30min, filtered with lens wiping paper, it is final
To the spore suspension of no count.Blood counting chamber is taken out, all spores recorded in internal 400 small lattice under the microscope are placed
Subnumber.
Total spore quantity of made spore suspension is finally calculated, dilution is configured to 1.0 × 107A mL−1Spore
Sub- suspension.
(2) result, which is checked and approved, examines.
The spore suspension and the spore suspension after dilution that undiluted concentration is added dropwise in glass slide.It observes under the microscope
Spore quantity on the spore suspension of undiluted concentration and the spore suspension glass slide after dilution, it is obvious poor to detect whether
It is different.
Embodiment 4
With beauveria bassianaBeauveria bassianaFungal bacterial strain BBXDCSFJ03 carries out biological control effect measuring.
The filter paper on culture dish middle berth is put into a little feed in each ware, by test worm trident maple material bark beetle (roundlet chest bark beetleEuwallacea fornicatus) (3, every ware) is put in glass ware, it is raised at room temperature.With toothpick by experimental bacteria (bacterium solution
Concentration 1.0 × 107A mL−1Spore suspension) it chooses with polypide, then the feed of picking part bacterium and feeding mixes, with
Just wedging together when worm feeding carries out indoor observation.In addition, each processing is heavy not choose the test worm into bacterium as control (CK)
It is 3 times multiple, 1-7 days after processing, polypide morbidity and death condition are periodically observed and recorded daily.
It is transferred to constant temperature incubation 8d in incubator, unpacks within every 24 hours and takes out a glass ware, record dead material bark beetle quantity.And
Dead material bark beetle is chosen into moisturizing culture, has checked whether that fungi grows, has contrasted the original for leading to the bark beetle death of trident maple material
Cause.Calculate material bark beetle cumulative mortality.Test is in triplicate.
Embodiment 5
Beauveria bassianaBeauveria bassianaFungal bacterial strain BBXDCSFJ03 carries out biological control effect measuring.
The filter paper on culture dish middle berth is put into a little feed in each ware, by test worm (trident maple material bark beetleXyleborus
Sp. it) is put in glass ware (3, every ware), is raised at room temperature.With toothpick by experimental bacteria (bacterial concentration 1.0 × 107A mL−1
Spore suspension) it chooses with polypide, then the feed of picking part bacterium and feeding mixes, so as to wedging together when worm feeding,
Carry out indoor observation.In addition, each processing is repeated 3 times, 1-7 days after processing, often not choose the test worm into bacterium as control (CK)
Its timing observes and records polypide morbidity and death condition.
It is transferred to constant temperature incubation 8d in incubator, unpacks within every 24 hours and takes out a glass ware, record dead material bark beetle quantity.And
Dead material bark beetle is chosen into moisturizing culture, has checked whether that fungi grows, has contrasted the original for leading to the bark beetle death of trident maple material
Cause.Calculate material bark beetle cumulative mortality.Test is in triplicate.
Embodiment 6
Beauveria bassianaBeauveria bassianaFungal bacterial strain BBXDCSFJ03 carries out true branch and sprays biological control.
1, conidial method is prepared according to step (1) in above-described embodiment 2, by beauveria bassianaBeauveria bassianaFungal bacterial strain BBXDCSFJ03 conidium is added in the Tween-80 solution of 0 .05%, by following concentration or method
Experimental group and control group are set:
1.0×106A mL−1Spore suspension, 1.0 × 107A mL−1Spore suspension, blank control: clear water.
30 branches of each processing spray the beauveria bassiana of various concentration respectivelyBeauveria bassianaFungal bacterial strain
The conidial suspension of BBXDCSFJ03, range estimation branch is completely drenched, stops sprinkling.
After biological control handles 15d, deadwood item health part is peeled off go back to, checks and counts the dead worm of triangle of death maple material bark beetle
Quantity.
Embodiment 7
With beauveria bassianaBeauveria bassianaThe analysis of fungal bacterial strain BBXDCSFJ03 biological control effect data.
In laboratory, trident maple material bark beetle is subjected to spray-on process processing in different concentration spore suspensions, continuously
The death rate of 10-15d record trident maple material bark beetle；Branch is tapped, so that material bark beetle is climbed out of branch surface, sprays various concentration respectively
Beauveria bassianaBeauveria bassianaFungal bacterial strain BBXDCSFJ03 conidial suspension is soft with SPSS 22.0
The lethality of each concentration of part analytical calculation.Test data is handled using 22.0 software of SPSS, carries out significance difference analysis.
Calculation formula is as follows:
Borer population × 100% before the death rate (%)=death borer population/processing
Corrected mortality (%)=(the processing death rate-control death rate)/(1- compares the death rate) × 100%
The results show that beauveria bassiana after spray-on process processing 10-15dBeauveria bassianaFungal bacterial strain BBXDCSFJ03
Bacterial strain conidium is to material bark beetle virulence effect:
1.0×106A mL−1Spore suspension, 1.0 × 107A mL−1Spore suspension concentration respectively corresponds corrected mortality
For 67.05% and 70.55%.As shown in Table 1.The result shows that beauveria bassianaBeauveria bassianaFungal bacterial strain
BBXDCSFJ03 is to trident maple material bark beetle (roundlet chest bark beetleEuwallacea fornicatus) lethal effect it is very fast.
The beauveria bassiana of 1 various concentration of tableBeauveria bassianaFungal bacterial strain BBXDCSFJ03 bacterial strain spore is outstanding
Toxicity test of the supernatant liquid to material bark beetle
Note: different letters are indicated in 0.05 horizontal upper significant difference after data
Regression analysis is carried out to conidium concentration and trident maple material bark beetle corrected mortality with 22.0 software of SPSS.Recurrence side
Journey is Y=11.125X+2.143.
Concentration X is converted using the logarithm that the truth of a matter is 10.
The results show that after spray-on process processing 10-15d, beauveria bassianaBeauveria bassianaFungal bacterial strain
Virulence effect of the BBXDCSFJ03 bacterial strain conidium to trident maple material bark beetle entirety:
Bacterial strain is to the field drug effect virulence of trident maple material bark beetle, and 1.0 × 106A mL−1Spore suspension, 1.0 × 107A mL−1It is 212 and 284 that spore suspension concentration, which respectively corresponds trident maple material bark beetle death toll,.The result shows that beauveria bassianaBeauveria bassianaFungal bacterial strain BBXDCSFJ03 can be to laboratory rearing trident maple material bark beetle and time withered trident maple
Material bark beetle in branch causes to concentrate mortality.Lethal speed is fast.
Claims (3)
1. one plant of Strain of Beauveria bassiana, it is characterised in that the Strain of Beauveria bassiana classification naming isBeauveria bassiana, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation day:
On December 28th, 2018, deposit number: CGMCC No.17068, the beauveria bassiana belong to mycota, Deuteromycotina, silk
Spore guiding principle, Moniliales, Moniliaceae, Beauveria.
2. a kind of application of Strain of Beauveria bassiana described in claim 1, it is characterised in that the Strain of Beauveria bassiana
Application in preparation biological control trident maple material bark beetle biological agent.
3. the application of Strain of Beauveria bassiana according to claim 2, it is characterised in that the Strain of Beauveria bassiana
Preparation biological control trident maple material bark beetle biological agent includes bacterial strain activation and numerous, spore suspension the preparation of expansion and biocontrol effect
Determination step specifically includes:
A, bacterial strain activation and expansion are numerous: the Strain of Beauveria bassiana being inoculated into activation and is expanded on breeding culture medium, in 20 ~ 30 DEG C
Lower culture obtains activation for 10 ~ 20 days and expands numerous production spore bacterial strain；
B, the preparation of spore suspension: by activation and expand in numerous production spore strain inoculated to PDA culture medium, the constant temperature at 20 ~ 30 DEG C
Culture 10 ~ 20 days rinses the spore in culture dish with the aseptic aqueous solution of addition 0.05%Tween-80, by the bacterium in culture dish
Liquid and spore all pour into cup and do not stop to shake 30min, are filtered with lens wiping paper, finally obtain the spore suspension of no count,
It places under the microscope, blood counting chamber counts, and records all spore counts in internal 400 small lattice, finally calculates made
Spore suspension total spore quantity, dilution be configured to various concentration gradient spore suspension；
C, biocontrol effect measures: the filter paper on culture dish middle berth, a little feed is put into each ware, by test worm trident maple material bark beetle
(roundlet chest bark beetleEuwallacea fornicatus) (3, every ware) is put in ware, it is raised at room temperature, it will be real with toothpick
It tests bacterium to choose with polypide, then the feed of picking part bacterium and feeding mixes, so as to wedging together when worm feeding, carry out indoor
Observation；In addition, each processing is repeated 3 times not choose the test worm into bacterium as control (CK), 1-7 days after processing, timing is seen daily
It examines and records polypide morbidity and death condition.
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| CN115838637A (en) * | 2022-10-24 | 2023-03-24 | 中国科学院东北地理与农业生态研究所 | Entomogenous fungus and fermentation method and application thereof |
| CN115838637B (en) * | 2022-10-24 | 2024-03-15 | 中国科学院东北地理与农业生态研究所 | Entomogenous fungi and fermentation method and application thereof |
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