CN108676734A - One plant of insect pathogenic fungus and its application - Google Patents
One plant of insect pathogenic fungus and its application Download PDFInfo
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- CN108676734A CN108676734A CN201811021179.3A CN201811021179A CN108676734A CN 108676734 A CN108676734 A CN 108676734A CN 201811021179 A CN201811021179 A CN 201811021179A CN 108676734 A CN108676734 A CN 108676734A
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Abstract
One plant of insect pathogenic fungus and its application, the present invention relates to one plant of insect pathogenic fungus and its applications.The present invention provides one plant of insect pathogenic fungus and its applications.One plant of insect pathogenic fungus of the present invention is beauveria bassiana HUBB.No:08, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and preservation date is on June 11st, 2018, and deposit number is CGMCC No.15866.The present invention applies in potato disease control.Bacterial strain of the present invention has the ability of stronger inhibition of potato early epidemic germ mycelia growth, increases with the increase of volumetric concentration, the lasting period is long, and fungistatic effect is notable, and antibacterial efficiency is up to 81.73%, to illustrate bacterial strain HUBB.No:08 pair of target has good antagonism.The present invention is applied to technical field of environmental microorganism.
Description
Technical field
The present invention relates to one plant of insect pathogenic fungus and its applications.
Background technology
Target (Potato Early Blight) is one kind by sporulation (Alternaria solani
(Ell.et Mart.) Jones et Grout) caused by the disease more serious to potato hazard ratio, it is worldwide general
It is considered as the second largest potato disease for being only second to the late blight of potato in developing country all over generation.Early blight causes horse
Bell potato drops in production over a large area, and brings very serious economic loss.The disease has different degrees of generation in China potato producing region, and
In rising trend in recent years, the extent of injury in some areas has been no less than the late blight of potato.The cause of disease of target
Bacterium not only endangers potato, also infects the various crops such as tomato, eggplant, capsicum, beet and mustard category and causes early blight.Potato
Early blight occurs extensively in potato planting area of the world, is a kind of worldwide important potato disease.Early epidemic in recent years
Disease many potato producing regions frequently occur and it is on the rise, the potato underproduction, the serious time underproduction of disease can be caused reachable
50% or more.
Invention content
The present invention provides one plant of insect pathogenic fungus and its applications.
One plant of insect pathogenic fungus of the present invention is beauveria bassiana (Beauveria bassiana) HUBB.No:08, preservation
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is BeiChen West Road, Chaoyang District, BeiJing City 1
Number institute 3, preservation date is on June 11st, 2018, and deposit number is CGMCC No.15866.
The application of one plant of insect pathogenic fungus of the invention refers to the application in potato disease control.
The present invention is to obtain beauveria bassiana (Beauveria bassiana) HUBB.No in the following way:08：
Method is trapped using yellow meal worm, and the nascent type fungi of insect pathogenic fungus is isolated from the soil sample of Harbin City：
With isolated insect pathogenic fungus infection yellow meal worm (5 instar larvae), after polypide death, volumn concentration is used
For 75% alcohol impregnate 5s after, a small amount of white spore is scraped from polypide under aseptic condition on PDA solid mediums, as
It is cultivated 2-3 days in 28 DEG C of biochemical cultivation cases, picking white colony is birth type bacterium colony and is inoculated in improvement Martin culture solution
(potato cooking liquor 1000mL, glucose 20g, KH after filtering2PO4 3g、MgSO4·7H2O 1.5g, vitamin B1 8mg),
At 28 DEG C 180r/min shaken cultivations for 24 hours after, 1% be transferred in fermentation culture shaken cultivation by volume and ferment 36-48h
Afterwards, refrigerated centrifuge in centrifuge tube, takes supernatant, to obtain without fermented liquid.
The insect pathogenic fungus that separation screening obtains optics microscopically observation bacterium colony color and luster on nutrient medium is breast
White.Bacterium colony quality is powdery, bacterium colony is thin, intermediate fold, radial growth.Bacterium colony bottom is colourless or faint yellow.Bacterial strain produces
Spore cell is verticillate or Dan Sheng, conidiophore produce spore to top shaft type repeatedly, and as a result form geniculation there is small tooth to dash forward
Produce spore axis.Smooth conidiophore is in spike, and conidium is transparent, and wall is thin and is spherical shape, and surface is smooth.
Molecular Identification is carried out to the insect pathogenic fungus that separation screening obtains, carries out according to the following steps, extracts the total of bacterial strain
DNA carries out PCR amplification according to fungi universal primer by template of genomic DNA.Then plastic recovery kit is utilized to recycle pure
Change PCR product, is cloned, converted, be sequenced later.Sequencing result carries out homology pair with 16S rDNA sequences in Genbank
Than HUBB.No:The homology highest of 08 bacterial strain homology and Beauveria, 99% or more.By combine morphological features,
Molecular Identification result determines HUBB.No:08 bacterial strain is beauveria bassiana (Beauveria bassiana), is finally named as ball spore
Muscardine (Beauveria bassiana) HUBB.No:08.
Beauveria bassiana (B.bassiana) is one of the disinsection fungal for being widely used in biological control of insect pests both at home and abroad, can be had
Effect ground control population number, while other natural enemy insects and beneficial organism are not injured, bacteriostatic test plate of the present invention shows solid
Under body condition of culture, beauveria bassiana (Beauveria bassiana) HUBB.No of the present invention:08 also has stronger inhibition horse
The ability of bell potato early epidemic germ mycelia growth, increases with the increase of volumetric concentration, and the lasting period is long, and fungistatic effect is notable, suppression
Bacterium efficiency is up to 81.73%, to illustrate bacterial strain HUBB.No:08 pair of target has good antagonism.
The present invention effectively prevents target using the means of biological control, compares other traditional changes
Prevention is more environmentally-friendly, and non agricultural chemical residuum does not develop immunity to drugs.
Description of the drawings
Fig. 1 is beauveria bassiana HUBB.No:08 without thalline zymotic fluid improvement martin substratum on to potato early epidemic
Germ generates photo when antibacterial band 3 days；
Fig. 2 is beauveria bassiana HUBB.No:08 without thalline zymotic fluid improvement martin substratum on to potato early epidemic
Germ generates photo when antibacterial band 7 days.
Specific implementation mode
Specific implementation mode one：One plant of insect pathogenic fungus of present embodiment is beauveria bassiana (Beauveria
bassiana)HUBB.No:08, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address
It is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date is on June 11st, 2018, and deposit number is CGMCC
No.15866。
Present embodiment is to obtain beauveria bassiana (Beauveria bassiana) HUBB.No in the following way:08
's：
Method is trapped using yellow meal worm, and the nascent type fungi of insect pathogenic fungus is isolated from the soil sample of Harbin City：
With isolated insect pathogenic fungus infection yellow meal worm (5 instar larvae), after polypide death, volumn concentration is used
For 75% alcohol impregnate 5s after, a small amount of white spore is scraped from polypide under aseptic condition on PDA solid mediums, as
It is cultivated 2-3 days in 28 DEG C of biochemical cultivation cases, picking white colony is birth type bacterium colony and is inoculated in improvement Martin culture solution
(potato cooking liquor 1000mL, glucose 20g, KH after filtering2PO4 3g、MgSO4·7H2O 1.5g, vitamin B1 8mg),
At 28 DEG C 180r/min shaken cultivations for 24 hours after, 1% be transferred in fermentation culture shaken cultivation by volume and ferment 36-48h
Afterwards, refrigerated centrifuge in centrifuge tube, takes supernatant, to obtain without fermented liquid.
The insect pathogenic fungus that separation screening obtains optics microscopically observation bacterium colony color and luster on nutrient medium is breast
White.Bacterium colony quality is powdery, bacterium colony is thin, intermediate fold, radial growth.Bacterium colony bottom is colourless or faint yellow.Bacterial strain produces
Spore cell is verticillate or Dan Sheng, conidiophore produce spore to top shaft type repeatedly, and as a result form geniculation there is small tooth to dash forward
Produce spore axis.Smooth conidiophore is in spike, and conidium is transparent, and wall is thin and is spherical shape, and surface is smooth.
Molecular Identification is carried out to the insect pathogenic fungus that separation screening obtains, carries out according to the following steps, extracts the total of bacterial strain
DNA carries out PCR amplification according to fungi universal primer by template of genomic DNA.Then plastic recovery kit is utilized to recycle pure
Change PCR product, is cloned, converted, be sequenced later.Sequencing result carries out homology pair with 16S rDNA sequences in Genbank
Than HUBB.No:The homology highest of 08 bacterial strain homology and Beauveria, 99% or more.By combine morphological features,
Molecular Identification result determines HUBB.No:08 bacterial strain is beauveria bassiana (Beauveria bassiana), is finally named as ball spore
Muscardine (Beauveria bassiana) HUBB.No:08.
Beauveria bassiana (B.bassiana) is one of the disinsection fungal for being widely used in biological control of insect pests both at home and abroad, can be had
Effect ground control population number, while other natural enemy insects and beneficial organism are not injured, present embodiment bacteriostatic test plate shows
Under the conditions of solid culture, beauveria bassiana (Beauveria bassiana) HUBB.No:08 also has stronger inhibition Ma Ling
The ability of potato early epidemic germ mycelia growth, increases, the lasting period is long, and fungistatic effect is notable, antibacterial with the increase of volumetric concentration
Efficiency is up to 81.73%, to illustrate bacterial strain HUBB.No:08 pair of target has good antagonism.
Specific implementation mode two：The application of one plant of insect pathogenic fungus of present embodiment refers in potato disease control
Application.
Present embodiment effectively prevents target using the means of biological control, traditional compared to other
Chemical prevention it is more environmentally-friendly, non agricultural chemical residuum does not develop immunity to drugs.
Specific implementation mode three：Present embodiment is unlike specific implementation mode two：Insect pathogenic fungus is applied
On inhibition of potato early epidemic germ.Other are identical with embodiment two.
Specific implementation mode four：Present embodiment is unlike specific implementation mode two or three：Using with inhibition horse
The bacterium solution of the beauveria bassiana of bell potato early blight prevents target.Other are identical as specific implementation mode two or three.
Specific implementation mode five：Unlike one of present embodiment and specific implementation mode two to four：With inhibition horse
The bacterium solution preparation method of the beauveria bassiana of bell potato early blight is：By beauveria bassiana (Beauveria bassiana)
HUBB.No:08 is inoculated in fluid nutrient medium, and at 28 DEG C, under conditions of 180r/min, 36~48h of oscillation and fermentation cultivation is poured into
Refrigerated centrifuge is carried out in centrifuge tube, takes supernatant, obtains bacterium solution.Other are identical as one of specific implementation mode two to four.
Specific implementation mode six：Unlike one of present embodiment and specific implementation mode two to five：The liquid
Culture medium is improvement martin substratum, is formulated and is：The improvement martin substratum of 1L is by filtered 1000mL potatos
The KH of cooking liquor, the glucose of 20g, 3g2PO4, 1.5g MgSO4·7H2The vitamin B1 of O and 8mg forms.Other with it is specific
One of embodiment two to five is identical.
Specific implementation mode seven：Present embodiment present embodiment is to obtain beauveria bassiana in the following way
(Beauveria bassiana)HUBB.No:08：
Method is trapped using yellow meal worm, and the nascent type fungi of insect pathogenic fungus is isolated from the soil sample of Harbin City：
With isolated insect pathogenic fungus infection yellow meal worm (5 instar larvae), after polypide death, volumn concentration is used
For 75% alcohol impregnate 5s after, a small amount of white spore is scraped from polypide under aseptic condition on PDA solid mediums, as
It is cultivated 2-3 days in 28 DEG C of biochemical cultivation cases, picking white colony is birth type bacterium colony and is inoculated in improvement Martin culture solution
(potato cooking liquor 1000mL, glucose 20g, KH after filtering2PO4 3g、MgSO4·7H2O 1.5g, vitamin B1 8mg),
At 28 DEG C 180r/min shaken cultivations for 24 hours after, 1% be transferred in fermentation culture shaken cultivation by volume and ferment 36-48h
Afterwards, refrigerated centrifuge takes supernatant, to obtain without fermented liquid.
Specific implementation mode eight：Present embodiment beauveria bassiana Beauveria bassiana HUBB.No:08 mirror
It is fixed：
The insect pathogenic fungus that separation screening obtains optics microscopically observation bacterium colony color and luster on nutrient medium is breast
White.Bacterium colony quality is powdery, bacterium colony is thin, intermediate fold, radial growth.Bacterium colony bottom is colourless or faint yellow.Bacterial strain produces
Spore cell is verticillate or Dan Sheng, conidiophore produce spore to top shaft type repeatedly, and as a result form geniculation there is small tooth to dash forward
Produce spore axis.Smooth conidiophore is in spike, and conidium is transparent, and wall is thin and is spherical shape, and surface is smooth.
Molecular Identification is carried out to the insect pathogenic fungus that separation screening obtains, carries out according to the following steps, extracts the total of bacterial strain
DNA carries out PCR amplification according to fungi universal primer by template of genomic DNA.Then plastic recovery kit is utilized to recycle pure
Change PCR product, is cloned, converted, be sequenced later.DNA sequence dna such as SEQ ID NO：Shown in 1.In sequencing result and Genbank
16S rDNA sequences carry out homology alignment, HUBB.No:The homology highest of 08 bacterial strain homology and Beauveria, 99% with
On.By combining morphological features, Molecular Identification result to determine HUBB.No:08 bacterial strain is beauveria bassiana (Beauveria
Bassiana), finally it is named as beauveria bassiana (Beauveria bassiana) HUBB.No:08.
To the beauveria bassiana Beauveria bassiana HUBB.No of present embodiment:08 in inhibition of potato early epidemic
Functional authorization on germ：
Experiment 1：
Antagonism of the without fermented liquid to target is measured using gradient dilution method
1. early-stage preparations：The target bacterium that this laboratory preserves is inoculated in potato dextrose agar
(PDA) it is placed in 26 DEG C of constant incubators, the mycelia of culture is covered with spare when culture dish.
2. the preparation of potato dextrose agar (PDA)：Potato first cleans peeling, then weighs 200g potatos
It is cut into small pieces, adds boiling rotten (boiling 20~30 minutes, can be poked by glass bar), with eight layers of filtered through gauze, heating, then evidence
Actual experiment needs plus 15-20g agar, continues heating stirring mixing, after agar has dissolved, glucose is added, stirs evenly,
Moisture is supplied again after slightly cooling down to 1000 milliliters, is dispensed test tube or conical flask, is jumped a queue, wraps up, 20 minutes left sides of (115 DEG C) sterilizings
Test tube pendulum inclined-plane is taken out behind the right side or is shaken up, and is stored after cooling spare.
3. antagonism measures：Aseptically, the card punch for being 5mm with internal diameter punches in PDA culture medium, in hole
It is added without 60 μ L (being named as Bb08) of thalline zymotic fluid, away from inoculation target bacterium fungus block at the 4cm of the hole.Nothing is added
Bacterium water is control (being named as BEL), often handles 3 repetitions.Be placed in 26 DEG C of constant incubators, when compare bacterium colony grow to it is full when,
Measure antibacterial bandwidth.
4. interpretation of result：Beauveria bassiana HUBB.No after measured:08 have to target without thalline zymotic fluid is bright
Aobvious inhibiting effect, from the antibacterial band the 1st day of generation, until mycelia bypasses without fermented liquid inoculating hole, Antagonistic Fungi week after the 7th day
The average antibacterial band for generating 5mm wide is enclosed, the target bacterium close to Antagonistic Fungi side stops growing, to target
Antagonism result it is as illustrated in fig. 1 and 2, Fig. 1 be beauveria bassiana HUBB.No:08 is improveing Martin without thalline zymotic fluid
Photo when antibacterial band 3 days is generated on culture medium to target bacterium；Fig. 2 is beauveria bassiana HUBB.No:08 without thalline
Photo of the zymotic fluid when generating antibacterial band 7 days to target bacterium on improveing martin substratum.It can be with by Fig. 1 and 2
It obtains, the 7th day beauveria bassiana HUBB.No:The fungistatic effect of 08 pair of target bacterium is notable, illustrates that Insect Pathogenic is true
Bacterium beauveria bassiana HUBB.No:08 pair of target bacterium inhibits the lasting period longer.
Insect pathogenic fungus beauveria bassiana HUBB.No:08 have to target bacterium without thalline zymotic fluid is bright
Aobvious antagonism, and the lasting period is longer, can be good at the growth for inhibiting the target bacterium close to Antagonistic Fungi side,
Fungistatic effect is notable, and antibacterial efficiency is up to 81.73%.
Sequence table
<110>Harbin University
<120>One plant of insect pathogenic fungus and its application
<160> 1
<210> 1
<211> 577
<212> DNA
<213>Beauveria bassiana (Beauveria bassiana)
<400> 1
tagtcctacg cgcgatcagg atgaaacgta agctccagtg caagtcttca acccacaaaa 60
tgccgcaccg gcgcagcccc agggccacgc tcgacataat gacgcgtctc cagcggcgcc 120
tgcccggcgg tgaggtaaag tcccggccgc cacacgacgg ccaggggttg cggctggagg 180
ggttcccctc cagctcccaa ctttactgcg agcttgtccg tacgggcggt cttacgaccg 240
cccgcgttac acgcaagttt ctaagctact aagtgaccta agacgttaag tgtaatgaat 300
agcgcaaagc gacgcaagaa gtagctacgg tctcggttct ctaggcaaca actttcaaaa 360
ctaagtaaac aaaacggaac gccgcataag tcttctacga ccttatgttc tcaaactcca 420
ggggccgccc ggcgaccagg tcaggcgcag gcccgacccc gctcaggcgg cttcgttgct 480
atccatccaa gtgtcttccc aatccctcaa cttttgagcc attactaggg aggcgaccaa 540
gtggttgcct ctggaacaat gctaaaaaat gaagtat 577
Claims (6)
1. one plant of insect pathogenic fungus, it is characterised in that it is beauveria bassiana (Beauveria bassiana) HUBB.No:
08, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1, preservation date are on June 11st, 2018, and deposit number is CGMCC No.15866.
2. the application of one plant of insect pathogenic fungus as described in claim 1, it is characterised in that insect pathogenic fungus is in potato
Application in disease control.
3. the application of one plant of insect pathogenic fungus according to claim 2, it is characterised in that insect pathogenic fungus is applied
On inhibition of potato early epidemic germ.
4. the application of one plant of insect pathogenic fungus according to claim 2, it is characterised in that using with inhibition of potato
The bacterium solution of the beauveria bassiana of early blight prevents target.
5. the application of one plant of insect pathogenic fungus according to claim 2, it is characterised in that have inhibition of potato early epidemic
The bacterium solution preparation method of beauveria bassiana of disease is：By beauveria bassiana (Beauveria bassiana) HUBB.No:08 inoculation
In fluid nutrient medium, at 28 DEG C, under conditions of 180r/min, 36~48h of oscillation and fermentation cultivation, pour into centrifuge tube carry out it is cold
Freeze centrifugation, takes supernatant, obtain bacterium solution.
6. the application of one plant of insect pathogenic fungus according to claim 2, it is characterised in that the fluid nutrient medium is
Martin substratum is improved, is formulated and is：The improvement martin substratum of 1L be by filtered 1000mL potatos cooking liquor,
The glucose of 20g, the KH of 3g2PO4, 1.5g MgSO4·7H2The vitamin B1 of O and 8mg forms.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662830A (en) * | 2020-07-14 | 2020-09-15 | 哈尔滨学院 | Entomopathogenic fungi for inhibiting wheat damping-off and preparation method thereof |
| CN114456949A (en) * | 2022-01-13 | 2022-05-10 | 贵州民族大学 | Beauveria bassiana JSHA-MD912 and application thereof |
| CN117431175A (en) * | 2023-10-08 | 2024-01-23 | 云南师范大学 | Aquatic Lei Fusen bacterium ST172 and application thereof |
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| CN104073439A (en) * | 2013-12-06 | 2014-10-01 | 江西天人生态股份有限公司 | Beauveria bassiana(Bals.-Criv.)Vuill and application thereof |
| CN105918330A (en) * | 2016-05-24 | 2016-09-07 | 重庆大学 | New application of sirolimus in inhibition of plant alternaria solani |
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| CN105918330A (en) * | 2016-05-24 | 2016-09-07 | 重庆大学 | New application of sirolimus in inhibition of plant alternaria solani |
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| H REISENZEIN ET AL.: "Growth inhibiting effect of different isolates of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. to the plant parasitic fungi of the genera Fusarium, Armillaria and Rosellinia", 《PFLANZENSCHUTZ BERICHTE》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662830A (en) * | 2020-07-14 | 2020-09-15 | 哈尔滨学院 | Entomopathogenic fungi for inhibiting wheat damping-off and preparation method thereof |
| CN114456949A (en) * | 2022-01-13 | 2022-05-10 | 贵州民族大学 | Beauveria bassiana JSHA-MD912 and application thereof |
| CN114456949B (en) * | 2022-01-13 | 2023-06-16 | 贵州民族大学 | Beauveria bassiana JSHA-MD912 and application thereof |
| CN117431175A (en) * | 2023-10-08 | 2024-01-23 | 云南师范大学 | Aquatic Lei Fusen bacterium ST172 and application thereof |
| CN117431175B (en) * | 2023-10-08 | 2024-05-24 | 云南师范大学 | Aquatic Lei Fusen bacterium ST172 and application thereof |
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| Publication number | Publication date |
|---|---|
| CN108676734B (en) | 2021-08-06 |
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