CN110923154B - Beauveria bassiana bh001 for preventing and treating quercus pernyi pests and application thereof - Google Patents
Beauveria bassiana bh001 for preventing and treating quercus pernyi pests and application thereof Download PDFInfo
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- CN110923154B CN110923154B CN202010002494.2A CN202010002494A CN110923154B CN 110923154 B CN110923154 B CN 110923154B CN 202010002494 A CN202010002494 A CN 202010002494A CN 110923154 B CN110923154 B CN 110923154B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
Abstract
The invention discloses beauveria bassiana bh001 for preventing and treating quercus pernyi pests and application thereof. The invention provides Beauveria bassiana (Beauveria bassiana) bh001, which has a preservation number of CGMCC No.17975 in the common microorganism center of China Committee for culture Collection of microorganisms. The biocontrol strain Beauveria bassiana (Beauveria bassiana) bh001 provided by the invention can be used for efficiently preventing and controlling Quercus mongolicus elephants, has good biocontrol potential, provides a new resource for the biological prevention and control of forestry pests Quercus mongolicus elephants, and simultaneously provides help for the protection of Quercus mongolicus seed resources.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to beauveria bassiana bh001 for preventing and treating quercus pernyi pests and application thereof.
Background
The quercus mongolica forest is an important secondary forest resource in China, and quercus mongolica seed can be used for extracting starch, and has important significance on national food safety and energy safety if being used as feed or replacing corn to produce industrial ethanol. In recent years, over 60% of Quercus mongolicus seeds are damaged by Quercus mongolicus images, and huge economic loss is caused. At present, an effective and environment-friendly control means for oak elephant harm is lacked. Therefore, the oak elephant control technology based on pathogenic fungi is a great technical problem which needs to be solved urgently at present. At present, more than 170 entomopathogenic microorganism products are registered internationally, but at present, only 11 fungus insecticide products of 3 fungi such as beauveria bassiana, metarhizium anisopliae microsporum variant and locust variant are applied to the control of agricultural and forestry pests in China. The relative specificity of entomopathogenic microorganisms makes the search for indigenous superior strains of bacteria directed to specific forest pests an important step in the development of targeted and highly effective fungal pesticides. Research on the research of biological agents based on pathogenic fungi with high pathogenicity on oak elephant in the soil of Changbai mountain forest region and corresponding chemical prevention and control technology has not been reported.
Biological control is one of the hot spots of current disease control research. Biological control is to control the occurrence and harm of harmful organisms by using living organisms or metabolites thereof. Because the biological agent utilizes organisms or metabolites thereof which exist in nature, the biological agent is environment-friendly and safe to human and livestock, and harmful organisms are not easy to generate resistance. Biological control has natural advantages over chemical control. However, due to high production cost, short shelf life, great difficulty in using technology and the like, the production and application are relatively few. In recent years, the application of biological pesticides is effectively promoted by measures such as screening high-efficiency biocontrol strains, optimizing production processes and the like.
The application of beauveria bassiana to the prevention and treatment of forestry pests is one of the key points of the biological prevention and treatment research at present. However, the use of beauveria bassiana for preventing and treating quercus oak elephantiasis has not been reported.
Disclosure of Invention
The invention aims to provide Beauveria bassiana (Beauveria bassiana) bh001 for controlling oak elephant pests and application thereof.
In a first aspect, the invention claims Beauveria bassiana (Beauveria bassiana) bh 001.
The Beauveria bassiana (Beauveria bassiana) bh001 required to be protected by the invention is preserved in the China general microbiological culture Collection center in 2019, 6 months and 20 days, and the preservation number is CGMCC No. 17975.
In a second aspect, the invention claims a bacterial agent.
The active ingredient of the microbial inoculum claimed by the invention is Beauveria bassiana (Beauveria bassiana) bh 001.
In the microbial agent, the spore amount of Beauveria bassiana (Beauveria bassiana) bh001 may be 100(± 10) hundred million/g of the microbial agent.
Further, the microbial inoculum can be powder (the particle size is less than 35 meshes).
Furthermore, the microbial inoculum is conidium powder prepared by taking a mixture of wheat bran, rice chaff and corn grits (3:1:1) (mass ratio) as a culture medium, inoculating the Beauveria bassiana (Beauveria bassiana) bh001, and culturing, drying and crushing.
In a third aspect, the invention claims the use of Beauveria bassiana (Beauveria bassiana) bh001 as hereinbefore described or the inoculant in any one of:
(B1) preventing and controlling oak elephant;
(B2) preparing a product for preventing and controlling oak elephant.
In a fourth aspect, the invention claims a method of controlling oak elephant.
The method for controlling oak images as claimed by the invention can comprise the following steps: 2-3g of the microbial inoculum is sprayed on each square meter of the area to be controlled.
In a fifth aspect, the invention claims a method for forest application of the microbial inoculum described hereinbefore.
The forest application method of the microbial inoculum, which is claimed by the invention and is described in the foregoing, can comprise the following steps: 2-3g of the microbial inoculum is sprayed on each square meter of area to be controlled.
Further, in the fourth and fifth aspects, the fungicide is applied to the surface of the area to be controlled before oak elephant larva enters soil and overwinter
In a particular embodiment of the invention, the area to be controlled is in particular quercus mongolica forest.
The invention collects oak elephantiasis from Changbai mountain forest area, separates to obtain entomopathogenic fungus strain, screens out a biocontrol strain for efficiently preventing oak elephantiasis by measuring the pathogenicity of the separated strain, and identifies the biocontrol strain as beauveria bassiana by morphological and molecular biological analysis; the strain Beauveria bassiana bh001 has a remarkable prevention and treatment effect on Quercus elephantopus larvae, the prevention and treatment effect in an indoor test reaches 85.3%, and the prevention and treatment effect in a forest test reaches 70.1%. The biocontrol strain provided by the invention can be used for efficiently preventing oak elephants, has good biocontrol potential, provides a new resource for the biological prevention and control of forestry pests oak elephants, and simultaneously provides help for the protection of Mongolian oak seed resources.
Deposit description
The strain name: beauveria bassiana (balsamo) Vuillemin
Latin name: beauveria gossiana
According to the biological materials (strains): bh001
The preservation organization: china general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 6 months and 20 days in 2019
Registration number of the preservation center: CGMCC No.17975
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 acquisition and identification of Beauveria bassiana (Beauveria bassiana) bh001
1. Collection of entomopathogenic fungi
Collecting the dead oak weevils naturally infected with pathogenic fungi in the overwintering soil of the oak weevils in the Changbai mountain forest area of Jilin.
2. Isolation and purification of entomopathogenic fungi
The collected oak trunk infected with pathogenic fungi is subjected to surface disinfection by 95% ethanol, cut into tissue blocks with the size of 0.2mm by a knife, placed on a PDA plate culture medium, cultured at the constant temperature of 25 ℃, and the grown pathogenic fungi are purified to obtain pure strains.
3. Strain virulence determination
(1) Collecting wild oak elephant larva, and selecting healthy and active individual for test.
(2) Adding 20ml of 0.05% (volume percentage) TW-80 solution into 50ml triangular flasks, respectively, scraping cultured strain spore powder into each triangular flask with a spoon, shaking with vortex oscillator for 10min, measuring the initial concentration of each strain spore suspension under microscope with hemocytometer, and diluting to 1 × 108Spore. ml-1。
(3) Soaking Quercus acutissima larva in spore suspension for 15S by soaking method, and taking out with 0.05% (volume percentage content) TW-80 as blank control. Recording dead insect number and dead insect number, detecting infected dead insect, determining whether pathogenic strain is identical to original strain, correcting death rate, and measuring its LT50。
The results show that: the entomopathogenic fungi purified and separated in the step 2 have pathogenicity on Quercus larva, and one of the entomopathogenic fungi is bh001The strain has the highest toxicity to Quercus mongolicus larvae, and LT thereof50Is 5 d. In contrast, LT of the remaining strains50Are all larger than 6 d.
4. Identification of strains
(1) Observation of cell and colony morphology
Inoculating the screened high-toxicity strain (with the number being bh001) on a PDA (personal digital assistant) plate culture medium, culturing for 7d at the constant temperature of 25 ℃, observing colony characteristics, picking hyphae, observing hyphae and spore morphological characteristics under a microscope, and indicating that: the colony is white, the color of the matrix is light yellow, the colony is powdery, the texture is loose, the colony is thin, and the edge is radial. The hypha is long and thin, the conidiophores are single and are in a bottle shape, and the bottle shape of the conidiophores is obviously expanded from the base to the long and thin bottle shape and is different. Conidia grow on the zigzag peduncle, most of them are round balls, and the few are oval, colorless and single cells.
(2) Molecular biological identification
Extracting DNA of the strain bh001, amplifying ITS segments by using primers ITS1F and ITS4R, sequencing, and performing BLAST comparison to obtain an amplification product containing a sequence shown in SEQ ID No. 1.
ITS1F：5'-TCCGTAGGTGAACCTGCGG-3'；
ITS4R：5'-TCCTCCGCTTATTGATATGC-3'。
And determining the strain to be Beauveria bassiana through the sequence comparison result and combining the morphological identification result.
Beauveria bassiana (Beauveria bassiana) bh001 is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 6 and 20 days, and the preservation number is CGMCC No. 17975.
5. Determination of growth characteristics of high-virulence strains
And (3) carrying out basic biological characteristic measurement aiming at the Beauveria bassiana (Beauveria bassiana) bh001 screened by indoor toxicity measurement, culturing the strain on a PDA (personal digital assistant) culture medium, and observing and recording corresponding data and information.
(1) Determination of spore yield of strain
Pipette 500. mu.l of 1X 10 concentration into a pipette4Spore. ml-1Beauveria bassiana (Bea)The spore suspension of uvaria basssiana) bh001 was inoculated on a 90mm petri dish plate, the petri dish was rotated at a constant speed to uniformly distribute the liquid on the surface of the culture medium, and the culture was performed in a constant temperature incubator at 25 ℃ for 5 times. After 15 days of culture, 10ml of TW-80 solution with the concentration of 0.05% (volume percentage content) is added into the culture dish, the whole plate colony is scraped, the whole plate colony is blown by a pipette repeatedly, the bacterial liquid is transferred into a 50ml conical flask, and the operation is repeated until the volume is 20 ml. The Erlenmeyer flask is placed in a shaker to be sufficiently shaken to prepare spore suspension. Spore content was determined using a hemocytometer.
The results show that: the spore content is 9.2 × 109Spores/dish.
(2) Spore germination rate of strain
Preparing 1X 106Spore. ml-1The spore suspension of Beauveria bassiana (Beauveria bassiana) bh001 was inoculated into a triangular flask containing 20ml of PDA liquid medium, cultured at 25 ℃ under shaking at 150r/min for 12 hours, and then the spore germination was observed under a microscope. Spores with a germ tube length greater than or equal to the spore radius are considered germinated.
The results show that: the spore germination rate is 90.8%.
Example 2 prevention and treatment test of Beauveria bassiana (Beauveria bassiana) bh001 on Quercus acutissima
1. Indoor oak elephant prevention and control test
(1) Preparation of biocontrol bacterium
Beauveria bassiana (Beauveria bassiana) bh001 is taken as a production strain, a mixture of wheat bran, rice bran and corn grits (3:1:1) (mass ratio) is taken as a culture medium, and the culture medium is inoculated, cultured, dried, crushed and the like to prepare powder, the spore content is 100 +/-10 hundred million/g, and the fineness is that all the powder passes through a 35-mesh sieve.
(2) Quercus elephant larva preparation
Quercus elephant larvae are obtained from field collected Quercus mongolicus fruits, and prepared into 1m × 1m × 0.5m wooden box filled with soil. 30 replicates.
(3) Powder spray test
Spraying step (1), spraying 2-3g of beauveria bassiana powder per square meter, and then spraying 1m of beauveria bassiana powder on the surface layer of the soil2Placing 100 oak weevil larvae, spraying in contrast without graftingA culture powder of beauveria bassiana.
(4) Results survey
After 30d, the number of runt insects is investigated.
The results show that: the effect of preventing and treating oak elephant by beauveria bassiana bh001 reaches 85.3%.
2. Test for controlling oak elephant in forest
In a quercus mongolica forest, 30 sample plots of 2m multiplied by 2m are drawn according to the specific forest phase condition in the forest, each sample plot is covered by a rack net to prevent worm bodies from climbing out, and the contrast, the microbial inoculum dosage and the worm placing number are the same as those of an indoor control test. The experiment was started in the middle of 9 months, and the number of stiff insects was counted in the last 4 months of the second year. The control effect calculation formula is the same as the indoor control test calculation formula.
The results show that: the effect of preventing and treating oak elephant by beauveria bassiana bh001 reaches 70.1%.
Sequence listing
<110> university of North China
<120> beauveria bassiana bh001 for preventing and treating quercus pernyi pests and application thereof
<130> GNCLN200006
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 186
<212> DNA
<213> Beauveria bassiana
<400> 1
ttctgtgacc tacctatcgt tgcttcggcg gactcgcccc agcccggacg cggactggac 60
cagcggcccg ccggggacct caaactcttg tattccagca tcttctgaat acgccgcaag 120
gcaaaacaaa tgaatcaaaa ctttcaacaa cggatctctt ggctctggca tcgatgaaga 180
acgcag 186
Claims (10)
1. Beauveria bassiana basssiana bh001, and the preservation number of the Beauveria bassiana bh is CGMCC No.17975 in the common microorganism center of China Committee for culture Collection of microorganisms.
2. A microbial agent comprising Beauveria bassiana bh001 as defined in claim 1 as an active ingredient.
3. The microbial inoculum of claim 2, wherein: in the microbial inoculum, the spore amount of Beauveria bassiana bh001 of the Beauveria bassiana is 100 +/-10 hundred million/g of the microbial inoculum.
4. The microbial inoculum according to claim 2 or 3, characterized in that: the microbial inoculum is powder.
5. The microbial inoculum of claim 3, wherein: the microbial inoculum is conidium powder prepared by taking a mixture of wheat bran, rice chaff and corn grits as a culture medium, inoculating Beauveria bassiana bh001 to the Beauveria bassiana, and culturing, drying and crushing the Beauveria bassiana bh 001.
6. Use of the agent of Beauveria bassiana bh001 of claim 1 or any one of claims 2 to 5 in any one of:
(B1) preventing and controlling oak elephant;
(B2) preparing a product for preventing and controlling oak elephant.
7. A method for controlling oak elephant comprises the following steps: spraying 2-3g of the microbial inoculum according to any one of claims 3-5 per square meter of area to be controlled.
8. A method for the application of the fungicide according to any one of claims 3 to 5 in forest comprising the steps of: spraying 2-3g of the microbial inoculum according to any one of claims 3-5 per square meter of area to be controlled.
9. The method according to claim 7 or 8, characterized in that: the microbial inoculum is applied to the ground surface of the area to be controlled before oak weevil is planted into soil and overwintering.
10. The method according to claim 7 or 8, characterized in that: the area to be controlled is a quercus mongolica forest.
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| CN112725190B (en) * | 2020-12-14 | 2022-09-23 | 东北林业大学 | Beauveria bassiana and application thereof, and composite microbial agent and special disease-resistant insect-increasing compound fertilizer for Korean pine nut forest |
| CN114456814A (en) * | 2022-01-17 | 2022-05-10 | 聊城金余肥业有限公司 | Low-density water-retention insect-resistant saline-alkali soil conditioner and preparation method thereof |
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