CN109988718A - A kind of Paecilomyces lilacinus category fungal bacterial strain and its application - Google Patents

A kind of Paecilomyces lilacinus category fungal bacterial strain and its application Download PDF

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CN109988718A
CN109988718A CN201910398552.5A CN201910398552A CN109988718A CN 109988718 A CN109988718 A CN 109988718A CN 201910398552 A CN201910398552 A CN 201910398552A CN 109988718 A CN109988718 A CN 109988718A
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bacterial strain
paecilomyces lilacinus
spore
bark beetle
fungal bacterial
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伍建榕
马焕成
魏玉倩
洪英娣
马翔
刘丽
洪永生
龙娟
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Southwest Forestry University
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Abstract

The invention discloses a kind of Paecilomyces lilacinus category fungal bacterial strain and its application, the Paecilomyces lilacinus category fungal bacterial strain classification naming isPaecilomyces lilacinusDepositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation day: on December 28th, 2018, deposit number: CGMCC No.17069, the Paecilomyces lilacinus category fungi belong to fungus circle, Eumycota, Fungi Imperfecti, Hyphomycetales, Moniliaceae, paecilomyces.Using the application for the Paecilomyces lilacinus category fungal bacterial strain in preparation biological control trident maple material bark beetle biological agent.

Description

A kind of Paecilomyces lilacinus category fungal bacterial strain and its application
Technical field
The invention belongs to microorganisms to be separately cultured technical field, and in particular to a kind of Paecilomyces lilacinus category fungal bacterial strain and its Using.
Background technique
Harm of the important Landscape Tree Species trident maple in Kunming by bark beetle seriously destroys the construction of Kunming Scenery of gardens With cause huge economic loss, it is very urgent to the improvement of bark beetle.It is difficult to find since bark beetle body is small, lives in addition Hidden, host provides good barrier for its completion history of life, causes prevention and treatment difficult；And roundlet chest bark beetle is because of its host plant Numerous and widely distributed, difficulty of prevention and cure is bigger (Walgama R S. etc., 2012).Bark beetle is only within a period of time of new adult It is active in the external world, most times seek hidden life in life, mating, feeding usually under bark.It is showed according to bark beetle It is concealed that biological characteristics out can be seen that its harm has the characteristics that, 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 this big drawback of environmental pollution.And physical control method In radiation and the stifling prevention and treatment for being directed to quarantine bark beetle, be not suitable for the prevention and treatment of living body host plant, sick branch yet The physical method control worm effect of trimming is also not very good.And biological control method, it is the raising with country to environmental consciousness, at For the green control method gradually approved by people.Entomogenous fungi is the important factor and pest biology of pest population Natural regulation The important materials of prevention and treatment, using entomogenous fungi pest control, gradually 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 microorganism resource, this research is trained by separating from dead bark beetle adult Raise can be lethal to bark beetle illness bacterial strain as biocontrol microorganisms.The strain isolation is tried from dead bark beetle by tieback It tests, through statistical analysis, there is the bacterial strain of remarkable effect to material bark beetle lethal effect, be pale purple quasi- blueness through morphology and Molecular Identification Mould (Paecilomyces lilacinus), the effective biocontrol bacterial strain filtered out in this, as trident maple material bark beetle.
Summary of the invention
The first object of the present invention is to provide a kind of Paecilomyces lilacinus category fungal bacterial strain；Second be designed to provide it is described Paecilomyces lilacinus category fungal bacterial strain application.
Outer unless otherwise indicated, percentage employed in the present invention is percentage by volume.
The first object of the present invention be achieved in that the Paecilomyces lilacinus category fungal bacterial strain (Paecilomyces lilacinus) PLXDCSFJ04, be named as PLXDCSFJ04, be identified as Paecilomyces lilacinus (Paecilomyces lilacinus), it is isolated from the lethal trident maple material bark beetle of natural occurrence；Depositary institution: Chinese microorganism strain is protected It hides administration committee's common micro-organisms center (CGMCC), preservation day: on December 28th, 2018, deposit number: CGMCC No.17069, the Paecilomyces lilacinus category fungi belong to fungus circle, Eumycota, deuteromycetes, Hyphomycetales, Moniliaceae, intend Penicillium.
Paecilomyces lilacinus of the present invention (Paecilomyces lilacinus), be named as PLXDCSFJ04, be from Naturally it falls ill isolated on dead trident maple material bark beetle, on December 28th, 2018, is preserved in Chinese microorganism strain 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:17069.
The bacterial strain is good to the causative effect of trident maple material bark beetle, can penetrate trident maple material bark beetle body segment in a short time Place, 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 and collects mitogenetic spore in trident maple material bark beetle body surface later Son, tieback to trident maple material bark beetle.It is verified by Koch's Postulates, tieback bacterial strain conidium infects other health experiments with three Angle maple material bark beetle has same virulence lethal effect.
The bacterial strain is obtained by following specific steps:
A, trident maple material bark beetle acquires: acquiring back trident maple material bark beetle living worm body and nature that withered trident maple branch strips out health Die of illness polypide, 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 Polypide head, chest, abdomen different parts separate, and the different physical feeling of the trident maple material bark beetle after separating carefully is placed into In 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 Paecilomyces lilacinus (Paecilomyces lilacinus) fungal bacterial strain PLXDCSFJ04 and its anti-in biology The method for controlling trident maple material bark beetle, activation and expansion including bacterial strain are numerous, spore suspension production, cause the dead poison of trident maple material bark beetle Power detection, specifically includes the following steps:
A, bacterial strain activation with expansion it is numerous: by Paecilomyces lilacinus (Paecilomyces lilacinus) fungal bacterial strain PLXDCSFJ04 connects Kind to activation with expand on breeding culture medium, is generated in 25 DEG C of ± lower culture 7-15d to a large amount of conidiums, obtains activation and the numerous production of expansion Spore bacterial 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。
Activation is common PDA culture medium with breeding culture medium is expanded.
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.
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 beetleXyleborusSp. 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−1Spore suspension) it chooses with polypide, then the feed of picking part bacterium and feeding mixes, so as to worm Wedging together when feeding carries out indoor observation.In addition, each processing repeats 3 not choose the test worm into bacterium as control (CK) It is secondary, 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.
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.
The second object of the present invention is achieved in that the Paecilomyces lilacinus category fungal bacterial strain in preparation biological control Application in trident maple material bark beetle biological agent.
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 At bark beetle body segment, and parasitizes 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 biocontrol microorganisms of the present invention are from trident maple material bark beetle natural sense under natural environmental condition It dies of illness and dies separation and obtain, therefore, be used, can be as the method for trident maple material bark beetle biological control, and operating process Simply, control efficiency is good.
Detailed description of the invention
Fig. 1 be embodiment 1 in Paecilomyces lilacinus (Paecilomyces lilacinus) PLXDCSFJ04 trains in common PDA It supports on base, cultivates bacterial strain bacterium colony front form when 7d；
Fig. 2 be embodiment 1 in Paecilomyces lilacinus (Paecilomyces lilacinus) PLXDCSFJ04 conidium micrograph；
Fig. 3 be embodiment 5 in Paecilomyces lilacinus (Paecilomyces lilacinus) PLXDCSFJ04 conidiophore and point Raw spore；
Fig. 4 be embodiment 6 in Paecilomyces lilacinus (Paecilomyces lilacinus) PLXDCSFJ04 bacterial strain invades in limb Contaminate trident maple material bark beetle, the morphological feature of the microscopically observation of growth and development with trident maple material bark beetle；
Fig. 5 be embodiment 6 in Paecilomyces lilacinus (Paecilomyces lilacinus) PLXDCSFJ04 infects three in limb Angle maple material 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.
Paecilomyces lilacinus category fungal bacterial strain classification naming of the present invention isPaecilomyces lilacinus, preservation Unit: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation day: on December 28th, 2018, Deposit number: CGMCC No.17069, the Paecilomyces lilacinus category fungi belong to fungus circle, Eumycota, deuteromycetes, hyphomycete Mesh, Moniliaceae, paecilomyces.
The application of Paecilomyces lilacinus category fungal bacterial strain of the present invention is that the Paecilomyces lilacinus category fungal bacterial strain exists Prepare the application in biological control trident maple material bark beetle biological agent.
The Paecilomyces lilacinus category fungal bacterial strain preparation biological control trident maple material bark beetle biological agent includes that bacterial strain is living Change and expand numerous, spore suspension preparation and biocontrol effect determination step, specifically includes:
A, bacterial strain activation and expansion are numerous: the Paecilomyces lilacinus category fungal bacterial strain is inoculated into activation and is expanded on breeding culture medium, in It is cultivated at 20 ~ 30 DEG C and obtains within 10 ~ 20 days activation and expand 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
Paecilomyces lilacinus (Paecilomyces lilacinus) fungal bacterial strain PLXDCSFJ04 acquisition, identification and preservation.
(1) Paecilomyces lilacinus (Paecilomyces lilacinus) fungal bacterial strain PLXDCSFJ04 acquisition, identification.
Paecilomyces lilacinus of the present invention (Paecilomyces lilacinus) fungal bacterial strain PLXDCSFJ04 fungi, It falls ill from trident maple material bark beetle and obtains on polypide, trident maple material bark beetle is fallen ill, and to pick up from Kunming Green Lake Park, Heilongtang public for polypide Each Grand Duchy Triangle ID maple plantation such as garden, botanical garden, acquisition have back the trident maple branch of withered symptom to take back laboratory dissection, are good for The indoor raising of health, 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: being in powdery after Paecilomyces lilacinus strain inclined-plane culture, flat, surface does not have secretion.Plate It is larger to cultivate bacterium colony, round, protuberance, finer and close, bacterium colony surface is without secretion.Under the conditions of 28 DEG C, grow within general 3rd day White colony, the 4th day gradually at deeper color.Its conidiophore end is expanded, conidium be it is unicellular, in chain, Smoothly, colourless to yellow, there is the irregular branch of bottleneck shape or verticillate branch, bottle metulae portion is wider on falx.Verticillate branch It is made of cylindrical part, branch is bent to far from major axes orientation sometimes.
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 unit cell 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 PLXDCSFJ04 and Paecilomyces lilacinus fungal bacterial strain (Paecilomyces lilacinus) the similitude of 5.8S r DNA sequence dna reached 99%.Pass through sequence alignment and form Feature by bacterial strain PLXDCSFJ04 be accredited as Paecilomyces lilacinus fungal bacterial strain (Paecilomyces lilacinus).Institute of the present invention Its 5.8S r DNA sequence dna of the bacterial strain PLXDCSFJ04 stated is as follows:
(2) Paecilomyces lilacinus fungal bacterial strain (Paecilomyces lilacinus) PLXDCSFJ04 preservation.
By above-mentioned qualification result, confirmation bacterial strain PLXDCSFJ04 be Paecilomyces lilacinus fungal bacterial strain (Paecilomyces lilacinus) one kind, number PLXDCSFJ04 is preserved in Chinese microorganism strain preservation pipe on December 28th, 2018 Reason 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:17069.
Embodiment 2
Paecilomyces lilacinus fungal bacterial strain (Paecilomyces lilacinus) fungal bacterial strain PLXDCSFJ04 prepares biocontrol agent
(1) preparation of spore suspension.
Firstly, by Paecilomyces lilacinus fungal bacterial strain (Paecilomyces lilacinus) fungal bacterial strain PLXDCSFJ04 connects Kind in common PDA culture medium, 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
Paecilomyces lilacinus fungal bacterial strain (Paecilomyces lilacinus) fungal bacterial strain PLXDCSFJ04 prepares biocontrol agent.
(1) preparation of spore suspension.
Firstly, by Paecilomyces lilacinus fungal bacterial strain (Paecilomyces lilacinus) fungal bacterial strain PLXDCSFJ04 connects Kind in common PDA culture medium, 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 Paecilomyces lilacinus fungal bacterial strain (Paecilomyces lilacinus) to carry out biology anti-by fungal bacterial strain PLXDCSFJ04 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 5
Paecilomyces lilacinus fungal bacterial strain (Paecilomyces lilacinus) fungal bacterial strain PLXDCSFJ04 progress 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
Paecilomyces lilacinus fungal bacterial strain (Paecilomyces lilacinus) the true branch of fungal bacterial strain PLXDCSFJ04 progress Spray biological control.
1, conidial method is prepared according to step (1) in above-described embodiment 2, by Paecilomyces lilacinus fungal bacterial strain (Paecilomyces lilacinus) fungal bacterial strain PLXDCSFJ04 conidium be added 0 .05% Tween-80 solution in, By following concentration or method setting experimental group and control group:
1.0×106A mL−1Spore suspension, 1.0 × 107A mL−1Spore suspension, blank control: clear water.
30 branches of each processing, respectively spray various concentration Paecilomyces lilacinus fungal bacterial strain (Paecilomyces lilacinus) fungal bacterial strain PLXDCSFJ04 conidial suspension, 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 Paecilomyces lilacinus fungal bacterial strain (Paecilomyces lilacinus) fungal bacterial strain PLXDCSFJ04 biological control effect The analysis of fruit 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 Paecilomyces lilacinus fungal bacterial strain (Paecilomyces lilacinus) fungal bacterial strain PLXDCSFJ04 conidial suspension, With the lethality of 22.0 each concentration of software analytical calculation of SPSS.Test data is handled using 22.0 software of SPSS, carries out difference Significance 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 spray-on process processing 10-15d after Paecilomyces lilacinus fungal bacterial strain (Paecilomyces lilacinus) fungi Bacterial strain PLXDCSFJ04 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 Paecilomyces lilacinus fungal bacterial strain (Paecilomyces lilacinus) fungal bacterial strain PLXDCSFJ04 is very fast to the lethal effect of trident maple material bark beetle.
Toxicity test of the PLXDCSFJ04 bacterial strain spore suspension of 1 various concentration of table 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, Paecilomyces lilacinus fungal bacterial strain (Paecilomyces lilacinus) fungal bacterial strain PLXDCSFJ04 bacterial strain conidium is to the virulence effect of 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 Paecilomyces lilacinus fungi Bacterial strain (Paecilomyces lilacinus) fungal bacterial strain PLXDCSFJ04 can to laboratory rearing trident maple material bark beetle and The material bark beetle returned in withered trident maple branch causes to concentrate mortality.Lethal speed is fast.

Claims (3)

1. one plant of Paecilomyces lilacinus category fungal bacterial strain, it is characterised in that the Paecilomyces lilacinus category fungal bacterial strain classification naming isPaecilomyces lilacinus, 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.17069, the Paecilomyces lilacinus category fungi In fungus circle, Eumycota, Fungi Imperfecti, Hyphomycetales, Moniliaceae, paecilomyces.
2. a kind of application of Paecilomyces lilacinus category fungal bacterial strain described in claim 1, it is characterised in that the pale purple quasi- blueness The mould application for belonging to fungal bacterial strain in preparation biological control trident maple material bark beetle biological agent.
3. the application of Paecilomyces lilacinus category fungal bacterial strain according to claim 2, it is characterised in that the pale purple quasi- blueness Mould category fungal bacterial strain preparation biological control trident maple material bark beetle biological agent includes that bacterial strain activates and expands numerous, spore suspension match System and biocontrol effect determination step, specifically include:
A, bacterial strain activation and expansion are numerous: the Paecilomyces lilacinus category fungal bacterial strain is inoculated into activation and is expanded on breeding culture medium, in It is cultivated at 20 ~ 30 DEG C and obtains within 10 ~ 20 days activation and expand 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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