CN110438016A - A kind of Metarhizium anisopliae bacterial strain and its application - Google Patents
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Abstract
The invention discloses a kind of Metarhizium anisopliae bacterial strain and its application, a kind of Metarhizium anisopliae bacterial strain of the invention, the bacterial strain is Metarhizium anisopliae；The entitled Metarhizium anisopliae Metarhizium anisopliae JXAS-03 of preservation；It is preserved in China typical culture collection center, preservation address is Wuhan City, Hubei Province Hongshan District Bayi Road Wuhan University China typical culture collection center；Preservation date: on 08 14th, 2019；Deposit number: CCTCC M 2019626.Bacterial strain of the invention has stronger killing ability to mole cricket class pest, provides a new material for the prevention and treatment of mole cricket class pest.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of Metarhizium anisopliae bacterial strain and its application.
Background technique
Gryllotalpa spp Orthoptera Gryllotalpidae insect, popular name mole cricket, ground draw mqb, and common mainly has Oriental burmeister, North China mole cricket
Mqb etc..Mole cricket mainly passes through adult and saps a gallery under soil, Plant species eaten root and tender stem, and leads to the root and soil of plant indirectly
It is detached from, causes plant seedlings withered.Mole cricket is liked in soft, tunneling in wetland.Mole cricket is omnivorous insect, several
All crops, especially crop seeding and seedling raising phase are endangered, nursery failure or the underproduction are often resulted in, are a kind of important subterranean pest-insects,
How to prevent and treat is a problem in the urgent need to address.
The method of prevention and treatment mole cricket mainly has at present: (1) light trap: mole cricket has apparent phototaxis, using black light lamp,
Larger in relative humidity, wind speed is small, no moonlight, the sultry dusk rained fastly, can trap a large amount of mole cricket；(2) horsehit is fresh
Grass trapping: the organic matters compost such as mole cricket team horsehit has very strong taxis, stacks ground or organic matter place abundant, mole cricket in muck
Mqb is just clustered in this, is also often a common method in mole cricket prevention and treatment using muck plus poison trapping, such as Lv Jie patent: one
(the patent No.: 201310583677.8) described in the formula of the organic fertilizer of kind prevention mole cricket；(3) chemical pesticide period poison bait:
Mole cricket has stronger chemotaxis, and to half-mature millet is boiled, soya-bean cake or wheat bran have very strong hobby, utilize these materials
Poison is smeared, mole cricket can be trapped and killed,；(4) Extracts from Plant Recourses is trapped and killed: as yellow big patent (patent No.:
201711094609.X；A kind of mole cricket killing agent being not likely to produce drug resistance) described in, Li Yong pivot stores, and the extracts such as borneol are mixed
Close mole cricket insecticide made of object；Biological control: currently without using bacterium, the natural pathogenic microorganism of the mole crickets such as fungi or virus
The report for preventing and treating mole cricket.Physical method prevention and treatment, not only needs to put into a large amount of black light lamp, higher cost, and effect is general.Chemistry
Pesticide poison bait, control efficiency is pretty good, but be easy to cause environmental pollution, and endangers to the health care belt of humans and animals.Separation identification
There is higher killing ability to mole cricket, and to the natural pathogenic microorganism of person poultry harmless, biological control is carried out to mole cricket, is one
Good selection.
Summary of the invention
The object of the present invention is to provide the Metarhizium anisopliae bacterial strains that a kind of pair of mole cricket class pest has stronger infection ability
JXAS-03 and its application in insect biological control, especially mole cricket biological control.
Technical scheme is as follows: a kind of Metarhizium anisopliae bacterial strain of the invention, and the bacterial strain is chafer
Green muscardine fungus；The entitled Metarhizium anisopliae Metarhizium anisopliae JXAS-03 of preservation；It is preserved in Chinese Typical Representative training
Object collection is supported, preservation address is in Wuhan City, Hubei Province Hongshan District Bayi Road Wuhan University China typical culture collection
The heart；Preservation date: on 08 14th, 2019；Deposit number: CCTCC M2019626.
Further, for Metarhizium anisopliae JXAS-03 on PDA plate, initial stage vegetative growth stage is white, small-sized silk
Shape fungi, radially toward outgrowth, the later period, which produces spore stage thallus, becomes green or yellow green, and the most central idol of bacterium colony is dotted
White.
Further, the ITS sequence of the Metarhizium anisopliae is as shown in SEQ ID NO:1.
The Metarhizium anisopliae microbial inoculum of Metarhizium anisopliae preparation of the present invention.
Further, active constituent is at least one of following (a) (b) (c):
(a) fermentation culture medium or spore suspension of Metarhizium anisopliae described in claim 1；
(b) the ultrasound cracking supernatant of claim 1 gained Metarhizium anisopliae cell；
(c) the ultrasound cracking precipitating of claim 1 gained Metarhizium anisopliae cell.
The preparation method of Metarhizium anisopliae microbial inoculum of the present invention, includes the following steps:
(1) on the PDA plate that bacterial strain single bacterium is fallen within to the chloramphenicol containing 25 μ g/ml, temperature is 25-30 DEG C, cultivates 3-14
It rinses spore with 0.05% Tween-80, muscardine spore suspension microbial inoculum is made until generating green spores；
(2) it or by the single colonie picking in (1), is inoculated into the LB culture solution of the chloramphenicol containing 25 μ g/ml, temperature is
25-30 DEG C, stirring rate 100-250rpm, shaking table culture 3-10 days, the fermentation culture medium of the green muscardine fungus is prepared；
(3) by after the fermentation culture medium ultrasonication in (2), 3000-5000rpm/min is centrifuged lysate, and precipitating is
Green muscardine fungus cell ultrasound cracking precipitating；Supernatant is green muscardine fungus ultrasound cracking supernatant.
Metarhizium anisopliae bacterial strain of the present invention is preparing the application in biological insecticides.
The utility model has the advantages that bacterial strain of the invention has stronger killing ability to mole cricket class pest, and can be in mole cricket population
Epidemic disease is caused, provides a new material for the prevention and treatment of mole cricket class pest.
The invention has the following advantages that
(1) Metarhizium anisopliae of the present invention is separated from the Oriental burmeister bombys batryticatus of natural infection death, should
Bacterial strain is that mole cricket lives and infects in the natural environment, has stronger virulence to mole cricket.
(2) present invention is needed compared to period poison bait method etc. using chemical pesticide, and Metarhizium anisopliae is right to person poultry harmless
Plant growth does not also damage, and is a kind of environmentally friendly biological insecticides, agrees with very much China's Ecological Civilization Construction, green
The requirement of agricultural development.
Detailed description of the invention
Fig. 1 is the bombys batryticatus phenotypic map that Oriental burmeister of the invention infects Metarhizium anisopliae death；
Fig. 2 is Metarhizium anisopliae colonial morphology figure of the invention；
Fig. 3 is Metarhizium anisopliae JXAS-03ITS sequence comparison result figure in ncbi database of the invention.
Specific embodiment
The present invention is further illustrated by the following examples.It should be understood that these embodiments are explainations of the invention
And citing, and the range that the invention is not limited in any way.
Embodiment 1
A kind of Metarhizium anisopliae bacterial strain of the invention, the bacterial strain are Metarhizium anisopliae；The entitled cockchafer of preservation
Sub- green muscardine fungus Metarhizium anisopliae JXAS-03；It is preserved in China typical culture collection center, preservation address
It is Wuhan City, Hubei Province Hongshan District Bayi Road Wuhan University China typical culture collection center；Preservation date: 08 month 2019
14 days；Deposit number: CCTCC M 2019626.
For Metarhizium anisopliae JXAS-03 on PDA plate, initial stage vegetative growth stage is white, and small-sized filamentous fungi is in
Radial past outgrowth, the later period, which produces spore stage thallus, becomes green or yellow green, and the most central idol of bacterium colony is dotted white.
The nucleotide sequence of the Metarhizium anisopliae is as shown in SEQ ID NO:1.
The Metarhizium anisopliae microbial inoculum of Metarhizium anisopliae preparation of the present invention.Its active constituent is following (a) (b)
At least one of (c):
(a) fermentation culture medium or spore suspension of Metarhizium anisopliae described in claim 1；
(b) the ultrasound cracking supernatant of claim 1 gained Metarhizium anisopliae cell；
(c) the ultrasound cracking precipitating of claim 1 gained Metarhizium anisopliae cell.
The preparation method of Metarhizium anisopliae microbial inoculum of the present invention, includes the following steps:,
(1) on the PDA plate that bacterial strain single bacterium is fallen within to the chloramphenicol containing 25 μ g/ml, temperature is 30 DEG C, is cultivated 3 days, until
Green spores are generated, spore is rinsed with 0.05% Tween-80, muscardine spore suspension microbial inoculum is made；
(2) it or by the single colonie picking in (1), is inoculated into the LB culture solution of the chloramphenicol containing 25 μ g/ml, temperature 30
DEG C, shaking table culture 10 days, the fermentation culture medium of the green muscardine fungus was prepared in stirring rate 250rpm；
(3) by after the fermentation culture medium ultrasonication in (2), 5000rpm/min is centrifuged lysate, and precipitating is green muscardine fungus
Cell ultrasound cracking precipitating；Supernatant is green muscardine fungus ultrasound cracking supernatant.
Metarhizium anisopliae bacterial strain of the present invention is preparing the application in biological insecticides.
Embodiment 2
Embodiment 2 the difference from embodiment 1 is that: the preparation method of Metarhizium anisopliae microbial inoculum of the present invention, including
Following steps:
In step (1), on PDA plate that bacterial strain single bacterium is fallen within to the chloramphenicol containing 25 μ g/ml, temperature is 25 DEG C, training
It supports 14 days, until generating green spores, rinses spore with 0.05% Tween-80, muscardine spore suspension microbial inoculum is made；
In step (2), or by the single colonie picking in (1), it is inoculated into the LB culture solution of the chloramphenicol containing 25 μ g/ml
In, temperature is 25 DEG C, stirring rate 100rpm, and shaking table culture 3 days, the fermentation culture medium of the green muscardine fungus is prepared；
In step (3), after the fermentation culture medium ultrasonication in (2), 3000rpm/min is centrifuged lysate, precipitating
As green muscardine fungus cell ultrasound cracking precipitating；Supernatant is green muscardine fungus ultrasound cracking supernatant.
Embodiment 3
Embodiment 3 the difference from embodiment 1 is that: the preparation method of Metarhizium anisopliae microbial inoculum of the present invention, including
Following steps:
In step (1), on PDA plate that bacterial strain single bacterium is fallen within to the chloramphenicol containing 25 μ g/ml, temperature is 28 DEG C, training
It supports 10 days, until generating green spores, rinses spore with 0.05% Tween-80, muscardine spore suspension microbial inoculum is made；
In step (2), or by the single colonie picking in (1), it is inoculated into the LB culture solution of the chloramphenicol containing 25 μ g/ml
In, temperature is 28 DEG C, stirring rate 150rpm, and shaking table culture 8 days, the fermentation culture medium of the green muscardine fungus is prepared；
In step (3), after the fermentation culture medium ultrasonication in (2), 4000rpm/min is centrifuged lysate, precipitating
As green muscardine fungus cell ultrasound cracking precipitating；Supernatant is green muscardine fungus ultrasound cracking supernatant.
Test example 1
The separation of 1 Metarhizium anisopliae is identified
Fig. 1 is the bombys batryticatus phenotypic map that Oriental burmeister of the invention infects Metarhizium anisopliae death.Note: Oriental burmeister is golden
After the infection of tortoise green muscardine fungus, bombys batryticatus extremity portion place greening is visible from white hypha body longer in vivo elsewhere.Figure
Scale on 1 is scale bar.
Oriental burmeister was collected in Yichun City Yifeng County in 2018, and took back laboratory and carry out separation identification research.First
Bombys batryticatus sample take pictures with camera to save picture；Later, with the tweezers of disinfection, before one for removing Oriental burmeister sample
Limb carries out 3 cleanings with aqua sterilisa, removes residual ethanol solution after ethanol solution surface sterilization 15 seconds of 75%；With sterilized
After the mortar of bacterium grinds the forelimb, after adding 1 milliliter of water and mixing, 100 microlitres of lapping liquid coating PDA plates is taken, are placed in 27
Degree Celsius, it is cultivated in the constant temperature and humidity incubator of relative humidity 80%.After growing clone, single bacterium is picked up with oese and is fallen within newly
Culture medium flat plate on cross purifying, continuous purification several times after, with tweezers from monoclonal clamping parts mycelium lysate
After cracking (Lysis Buffer for Microorganism to Direct PCR), takes part lysate as template, use
ITS1/ITS4 primer pair is expanded, and PCR product is served the raw work in sea and is sequenced.ITS sequence is as shown in Figure 3.ITS sequence exists
It is found after being compared on NCBI BLAST database, it is the most similar as shown in table 1 to Metarhizium anisopliae, therefore be by this Strain Designation
Metarhizium anisopliae JXAS-03 strain.The entitled Metarhizium anisopliae Metarhizium anisopliae JXAS-03 of preservation；
It is preserved in China typical culture collection center, preservation address is Wuhan City, Hubei Province Hongshan District China, Bayi Road Wuhan University allusion quotation
Type culture collection；Preservation date: on 08 14th, 2019；Deposit number: CCTCC M 2019626.
The form of 2 Metarhizium anisopliaes
Fig. 2 is Metarhizium anisopliae colonial morphology figure of the invention；Metarhizium anisopliae JXAS-03 is on PDA plate, just
Phase vegetative growth stage is white, small-sized filamentous fungi, radially toward outgrowth, the later period produce spore stage thallus become green or
Yellow green, the most central idol of bacterium colony are dotted white.
Fig. 3 is Metarhizium anisopliae JXAS-03ITS sequence comparison result figure in ncbi database of the invention.
>ITS sequences CCCTTCCCGTACGGGGAACCTGCGGAGGGATCATTACCGAGTTATCCAACTCC
CATACCTTTAATTGTTGCTTCGGCGGGACTTCGCGCCCGCCGGGGACCCAAACCTTCTGAATTTTTTAATAAGTATC
TTCTGAGTGGTTTAAAAAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAG
CGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGTCAGTA
TTCTGGCGGGCATGCCTGTTCGAGCGTCATTACGCCCCTCAAGTCCCCTGTGGACTTGGTGTTGGGGATCGGCGAGG
CTGGTTTTCCAGCACAGCCGTCCCTTAAATTAATTGGCGGTCTCGCCGTGGCCCTCCTCTGCGCAGTAGTAAAGCAC
TCGCAACAGGAGCCCGGCGCGGTCCACTGCCGTAAAACCCCCCAACTTTTTATAGTTGACCTCGAATCAGGTAGGAC
TACCCGCTGAACTTAAGCATATCAATGGGCGGGGGAAGGGATGGC
Illustrate: sequence alignment result shows, all gold in the Metarhizium anisopliae and current database of this patent separation
Tortoise green muscardine fungus ITS different and most like HZ1 strains has 96.42% similitude.
Oriental burmeister killing ability measures in 3 rooms Metarhizium anisopliae bacterial strain JXAS-03
Metarhizium anisopliae bacterial strain JXAS-03 inoculation is lined on PDA plate, is placed in 27 degrees Celsius, relative humidity
It is cultivated in 80% constant temperature and humidity incubator, covers with entire plate to fungi, it, will with 0.05% Tween-80 solution washing plate
Fungal spore rinses, and is filtered to remove the fungus sporophore of bulk after collection conidium with filter paper.With cell count instrument meter
After number conidial suspension concentration, it is 1 × 10 that conidium, which is diluted to concentration with 0.05% Tween-80 solution,5, 1 × 106,
1×107Spore/mL solution.Three repetitions of each concentration, 10 mole cricket adults of each repeating groups.Spore suspension handles mole cricket
Mqb adult uses infusion method, i.e., each mole cricket adult is pressed into spore suspension liquid level or less with tweezers and maintained 3 seconds.
0.05% Tween-80 is as blank control.Treated, and mole cricket adult is put in 27 degrees Celsius, the constant temperature and humidity of relative humidity 80%
It is cultivated in incubator, takes out and record dead bombys batryticatus quantity daily, calculate the death rate of each concentration, data are as follows: chafer
Mole cricket killing ability test result is as shown in table 1 in the room Metarhizium Strains JXAS-03:
Table 1
| Spore suspension concentration (spore/mL) | Death rate % (Mean Death number/10) |
| 1*105 | 43.3% (4.33/10) |
| 1*106 | 60% (6/10) |
| 1*107 | 83.3% (8.33/10) |
As shown in table 1, Metarhizium anisopliae bacterial strain JXAS-03 of the invention is stronger to indoor mole cricket killing ability.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, the present invention
Claimed range is delineated by the appended claims, the specification and equivalents thereof from the appended claims.
Sequence table
<110>institute of microbiology, In Jiangxi Science institute
<120>a kind of Metarhizium anisopliae bacterial strain and its application
<130> 2019
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 560
<212> DNA
<213>artificial sequence (nucleotide sequence of Metarhizium anisopliae ITS)
<400> 1
cccttcccgt acggggaacc tgcggaggga tcattaccga gttatccaac tcccatacct 60
ttaattgttg cttcggcggg acttcgcgcc cgccggggac ccaaaccttc tgaatttttt 120
aataagtatc ttctgagtgg tttaaaaaaa atgaatcaaa actttcaaca acggatctct 180
tggttctggc atcgatgaag aacgcagcga aatgcgataa gtaatgtgaa ttgcagaatt 240
cagtgaatca tcgaatcttt gaacgcacat tgcgcccgtc agtattctgg cgggcatgcc 300
tgttcgagcg tcattacgcc cctcaagtcc cctgtggact tggtgttggg gatcggcgag 360
gctggttttc cagcacagcc gtcccttaaa ttaattggcg gtctcgccgt ggccctcctc 420
tgcgcagtag taaagcactc gcaacaggag cccggcgcgg tccactgccg taaaaccccc 480
caacttttta tagttgacct cgaatcaggt aggactaccc gctgaactta agcatatcaa 540
tgggcggggg aagggatggc 560
Claims (7)
1. a kind of Metarhizium anisopliae bacterial strain, it is characterised in that: the bacterial strain is Metarhizium anisopliae；The entitled cockchafer of preservation
Sub- green muscardine fungus Metarhizium anisopliae JXAS-03；It is preserved in China typical culture collection center, preservation address
It is Wuhan City, Hubei Province Hongshan District Bayi Road Wuhan University China typical culture collection center；Preservation date: 08 month 2019
14 days；Deposit number: CCTCC M 2019626.
2. Metarhizium anisopliae bacterial strain according to claim 1, it is characterised in that: Metarhizium anisopliae JXAS-03 is in PDA
On plate, initial stage vegetative growth stage is white, and small-sized filamentous fungi, radially toward outgrowth, the later period produces spore stage thallus
Become green or yellow green, the most central idol of bacterium colony is dotted white.
3. Metarhizium anisopliae bacterial strain according to claim 1, it is characterised in that: the ITS of the Metarhizium anisopliae
Sequence is as shown in SEQ ID NO:1.
4. the Metarhizium anisopliae microbial inoculum of Metarhizium anisopliae preparation described in claim 1.
5. Metarhizium anisopliae microbial inoculum according to claim 4, active constituent is at least one in following (a) (b) (c)
Kind:
(a) fermentation culture medium or spore suspension of Metarhizium anisopliae described in claim 1；
(b) the ultrasound cracking supernatant of claim 1 gained Metarhizium anisopliae cell；
(c) the ultrasound cracking precipitating of claim 1 gained Metarhizium anisopliae cell.
6. the preparation method of the Metarhizium anisopliae microbial inoculum of claim 4 or 5, it is characterised in that include the following steps:
(1) on the PDA plate that bacterial strain single bacterium is fallen within to the chloramphenicol containing 25 μ g/ml, temperature is 25-30 DEG C, culture 3-14 days, directly
To green spores are generated, spore is rinsed with 0.05% Tween-80, muscardine spore suspension microbial inoculum is made；
(2) it or by the single colonie picking in (1), is inoculated into the LB culture solution of the chloramphenicol containing 25 μ g/ml, temperature 25-30
DEG C, shaking table culture 3-10 days, the fermentation culture medium of the green muscardine fungus was prepared in stirring rate 100-250rpm；
(3) by after the fermentation culture medium ultrasonication in (2), 3000-5000rpm/min is centrifuged lysate, and precipitating is green deadlock
Bacterium cell ultrasound cracking precipitating；Supernatant is green muscardine fungus ultrasound cracking supernatant.
7. Metarhizium anisopliae bacterial strain described in claim 1 is preparing the application in biological insecticides.
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| CN112646735A (en) * | 2021-01-21 | 2021-04-13 | 慕恩（广州）生物科技有限公司 | Metarhizium anisopliae, microbial insecticide, preparation method and application |
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| CN107022355A (en) * | 2017-05-04 | 2017-08-08 | 扬州臻微生物技术有限公司 | A kind of biologic ferment soil conditioner and preparation method thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112646735A (en) * | 2021-01-21 | 2021-04-13 | 慕恩（广州）生物科技有限公司 | Metarhizium anisopliae, microbial insecticide, preparation method and application |
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