CN109337823A - Metarhizium anisopliae IPPMHBC-009 and its application - Google Patents
Metarhizium anisopliae IPPMHBC-009 and its application Download PDFInfo
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- CN109337823A CN109337823A CN201811181953.7A CN201811181953A CN109337823A CN 109337823 A CN109337823 A CN 109337823A CN 201811181953 A CN201811181953 A CN 201811181953A CN 109337823 A CN109337823 A CN 109337823A
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Abstract
The invention belongs to field of biotechnology, and in particular to a kind of Metarhizium anisopliae IPPMHBC-009 and its application.Metarhizium anisopliae (Metarhizium anisopliae) IPPMHBC-009, deposit number are CGMCC No.16000, have good LD50 time and bombys batryticatus rate for Oedaleus asiaticus B, Dasyhippus barbipes.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of Metarhizium anisopliae IPPMHBC-009 and its application.
Background technique
Locust is the disastrous insect of Orthoptera locust section, and the plague of locusts frequently occurs in China, causes to China's agricultural huge
Injury.Currently, China control grassland grasshopper harm means mainly use chemical prevention method, have quickly, efficiently, make
With it is flexible the advantages that, still, as people increasingly pay attention to the sustainable development of environmental economy, biological locust elimination method is developed
Come and applies in real work.Biology goes out locust method, especially green muscardine fungus, muscardine etc., has certain specificity, to people
It raises harmless, while also having many advantages, such as that free from environmental pollution, noresidue, pest will not develop drug resistance.
Although disinsection fungal, which prevents and treats locust, has certain control efficiency, fungi is knocked down, killing off the insect pests acts on slowly,
It needs just be worked after a period of time, is suitble to general generation time control and application, and in locust outbreak year part, or part
When regional serious generation, disinsection fungal cannot quickly reduce insect density in the short time.Meanwhile in production practices and application process
It was found that a degree of degeneration often occur in some bacterial strains, show as after several generations is manually cultivated, bacterium colony is unstable, aerial hyphae
The features such as excessive growth, sporulation quantity are reduced, to influence control efficiency.
Summary of the invention
It is knocked down to solve fungi existing in the prior art, killing off the insect pests acts on the problems such as slow, present invention offer one
Kind Metarhizium anisopliae IPPMHBC-009 and its application.
The purpose of the present invention is to provide a kind of Metarhizium anisopliae IPPMHBC-009.
A further object of the present invention is to provide the applications of Metarhizium anisopliae IPPMHBC-009.
A further object of the present invention is to provide the biological prevention and control agents containing Metarhizium anisopliae IPPMHBC-009.
A further object of the present invention is to provide the methods using Metarhizium anisopliae IPPMHBC-009 prevention and treatment locust.
Metarhizium anisopliae (Metarhizium anisopliae) IPPMHBC- of specific embodiment according to the present invention
The deposit number CGMCC No.16000 of 009 bacterial strain, the bacterial strain were stored in China General Microbiological bacterium on 08 02nd, 2018
Kind collection, collection address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
Spore can be made in Metarhizium anisopliae IPPMHBC-009 by the biological prevention and control agent of specific embodiment according to the present invention
Powder is directly applied, or is combined and is used with other chemicals.
The method of the prevention and treatment locust of specific embodiment according to the present invention, including sow or spray to field and contain green deadlock
The Agrotechnical formulation of bacterium IPPMHBC-009.
Metarhizium anisopliae IPPMHBC-009 of the invention is 3.87d, bombys batryticatus rate to the median lethal time of Dasyhippus barbipes
It is 91.67%, the median lethal time to Oedaleus asiaticus B is 3.55d, and bombys batryticatus rate is 93.33%, therefore, chafer of the invention
Green muscardine fungus IPPMHBC-009 is with good application prospect.
Detailed description of the invention
Fig. 1 shows 9 kinds of green muscardine fungus to the cumulative mortality curve of Dasyhippus barbipes 15d;
Fig. 2 shows 9 kinds of green muscardine fungus to the cumulative mortality curve of Oedaleus asiaticus B 15d.
The deposit number CGMCC of Metarhizium anisopliae (Metarhizium anisopliae) IPPMHBC-009 bacterial strain
No.16000 was stored in China General Microbiological Culture Collection Center, collection address: Beijing on 08 02nd, 2018
The Institute of Microorganism, Academia Sinica of institute of Chaoyang District North Star West Road 1.
Specific embodiment
Bacterium source of the present invention:
The present invention tests 9 kinds of green muscardine fungus (Metarhizium anisopliae) bacterial strain, numbers respectively are as follows:
①IPPBHC-5;
②IPPMHBC-009;
③IPPMHK-7;
④IPPM189;
⑤IPPM803;
⑥IPPM200614;
⑦IPPM2010-10;
⑧IPPB054;
⑨IPPMC384;
The source of test worm of the present invention:
1. Dasyhippus barbipes (Dasyhippus barbipes Fischer-Waldheim): 2~3 age nymphs of a locust, method of beating
It is collected in In Xilingol League In Inner Mongolia white clouds coulomb locust area.
2. Oedaleus asiaticus B (Oedaleus decorus asiaticus B.Bienko): 2~3 age nymphs of a locust, the method for beating are adopted
Combine in the In Xilingol League In Inner Mongolia west northern 7 kilometers of areas Chu Huang of black flag Bayan Gol.
The locust raising of acquisition feeds suitable fresh sheep's hay in the big frame in experiment station dedicated for supporting locust daily
Blade.For guarantee test effect, test the previous day Nature enemy.
The screening of 1 green muscardine fungus IPPMHBC-009 of embodiment
Method is trapped from the soil of collection using greater wax moth and traps Metarhizium Strains.By with the scrap rubber of disinfection volume it is box-packed enter confession
Soil sample is tried, every box is embedded to 1 greater wax moth, is repeated 3 times.Soil sample box is placed on 24~27 DEG C, after moisturizing traps 1 week, sorts out dead worm,
After cultivating 7~10 days in 25~26 DEG C of constant temperature, relative humidity 100, aimed strain, strain isolation culture are separated from bombys batryticatus
Base is PDAY.
The green muscardine fungus being separated to is inoculated on PDAY solid medium, culture to production spore, 0.1% tween water of conidia powder
It suspends, vortex oscillator shaken well, adjustment spore concentration is 1 × 108A/mL.1mL spore suspension is taken to be inoculated into 100mL
Cultural hypha base is produced, 28 DEG C, 200r/min shaking table shaken cultivation 3d to logarithmic phase are set.It is filtered to obtain mycelia with vacuum pump, takes 1g
Mycelia liquid nitrogen grinding to powder extracts genomic DNA.Using genomic DNA as template, primer amplified is designed
The ITS1-5.8SrRDNA-ITS2 sequence of IPPMHBC-009 bacterial strain.PCR product is separated through 1.5% agarose gel electrophoresis, is used
Plastic recovery kit recycles purpose segment, is cloned into pGEM-T Easy Vector carrier, conversion to Trans-T1Phage
In Resistant competent cell, transformant is screened by blue hickie on ampicillin plate, after PCR is detected, is surveyed
Sequence.The sequence of measurement is subjected to BLAST analysis in GenBank, determines that bacterial strain is Metarhizium Strains, and name as IPPMHBC-
009。
Bioassay of the 2. green muscardine fungus deep blue 09 of embodiment to Dasyhippus barbipes nymph of a locust's pathogenicity
Above-mentioned 9 kinds of Metarhizium Strains are measured to the virulence effect of locust using stomach toxicity method.
0.2g muscardine spore powder is weighed first to be added in 2.0g wheat bran, adds 0.1ml edible oil, is sufficiently stirred
It is even.Then it weighs the green muscardine fungus wheat bran bait formulation that 0.2g is prepared to be put into culture dish, is put into togerther cover glass plate together with culture dish
Worm basket, number are supported, then is put into 20 3 age Dasyhippus barbipes nymphs of a locust of the same size in feeding worm basket.Blank control group wheat bran bait formulation
In muscardine spore powder is not added.3 weights are arranged in totally 9 kinds of muscardine spore powder and a control CK, every kind of conidia powder for this experiment
It is multiple, in being raised in insectary.Bait formulation was taken out in second day, feeds suitable fresh sheep's hay daily later, and investigate every basket of nymph of a locust
Death condition, make a record respectively.
The dead nymph of a locust is sorted out, being lined in the culture dish of filter paper for reference numeral is put into, keeps certain humidity, be allowed to long
Bombys batryticatus out.Investigate every kind of bacterium after the nymph of a locust to be tested is all dead treated the bombys batryticatus rate of the nymph of a locust, it is every in 15 days after combination processing
The cumulative mortality and median lethal time (LT of day50) pick out to the preferable strain of locust lethal effect.
9 plants of Metarhizium Strains are shown in figure l, table 1 to the bioassay results of 3 age Dasyhippus barbipes nymph of a locust pathogenicities.
Comparison of the 19 kinds of green muscardine fungus of table to Dasyhippus barbipes pathogenicity
Analyzed by table 1 it is found that above-mentioned 9 kinds of strains testeds have to the nymph of a locust it is pathogenic, median lethal time 3.87d~
Between 8.83d, bombys batryticatus rate is between 43.33%~91.67%.Wherein the median lethal time of green muscardine fungus IPPMHBC-009 is
3.87d, bombys batryticatus rate is 91.67%, and the bacterial strains such as IPPM803, IPPM200614, IPPM2010-10 median lethal time is slightly long, stiff
Worm rate is lower.Therefore, Metarhizium Strains IPPMHBC-009 exists significant compared with Metarhizium Strains IPPM803, IPPM200614
Difference shows that bacterial strain IPPMHBC-009 has higher pathogenicity to Dasyhippus barbipes.
Biometric of 3. green muscardine fungus of embodiment to Oedaleus asiaticus B pathogenicity
9 plants of bacterial strains are measured to the power of curing the disease of Oedaleus asiaticus B, method of the measuring method with embodiment 2.
9 plants of Metarhizium Strains are shown in Fig. 2, table 2 to the bioassay results of Oedaleus asiaticus B pathogenicity.
Comparison of the 29 plants of green muscardine fungus of table to Oedaleus asiaticus B pathogenicity
Analyzed by table 2 it is found that strains tested is to the nymph of a locust have it is pathogenic, median lethal time between 3.55d~10.67d,
Bombys batryticatus rate is between 38.33%~93.33%, and wherein the median lethal time of green muscardine fungus IPPMHBC-009 is 3.55d, bombys batryticatus rate
It is 93.33%.Therefore, Metarhizium Strains IPPMHBC-009 exists aobvious compared with Metarhizium Strains IPPM803, IPPM200614
Difference is write, shows that bacterial strain IPPMHBC-009 has higher pathogenicity to Oedaleus asiaticus B.
4. green muscardine fungus different strains growth rate of embodiment and sporulation quantity measurement
Different Metarhizium Strains spores is diluted to identical 2.5x108Spore/gram, take 10ul to be transferred to PDAY respectively
On solid plate, plate is placed in 27 DEG C of incubators and is cultivated, since the 4th day, the colony diameter of measurement in every two days, as a result
It is shown in Table 3.
The growth rate of the different Metarhizium Strains of table 3 compares
As shown in table 3, the growth rate of green muscardine fungus IPPMHBC-009 is apparently higher than control strain IPPM189 and other 4
Green muscardine fungus wild strain.
Different Metarhizium Strains are transferred on PDAY solid plate, continuously cultivated for 5 generations indoors, is often commissioned to train and supports to 15 days
(about 10 days after production spore), measures its sporulation quantity, the results are shown in Table 4.
The sporulation quantity of the different Metarhizium Strains of table 4 compares
As shown in table 4, after culture mostly generation, IPPMHBC-009 still has preferable sporulation quantity, and it is steady to produce spore character
It is fixed, it is not significantly different with control strain.
Claims (5)
- Metarhizium anisopliae 1. (Metarhizium anisopliae) IPPMHBC-009, which is characterized in that the chafer is green The deposit number of stiff bacterium IPPMHBC-009 is CGMCC No.16000.
- 2. Metarhizium anisopliae (Metarhizium anisopliae) IPPMHBC-009 according to claim 1 is anti- Application in locust elimination worm.
- 3. Metarhizium anisopliae (Metarhizium anisopliae) IPPMHBC-009 according to claim 2 is anti- Application in locust elimination worm, which is characterized in that the locust includes Oedaleus asiaticus B and/or Dasyhippus barbipes.
- 4. a kind of locust biocontrol agent, which is characterized in that the locust biocontrol agent includes cockchafer described in claim 1 Sub- green muscardine fungus (Metarhizium anisopliae) IPPMHBC-009.
- 5. a kind of method for preventing and treating locust, which is characterized in that comprising green using chafer described in claim 1 in the method The step of stiff bacterium (Metarhizium anisopliae) IPPMHBC-009 prevention and treatment locust.
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
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CN109769861A (en) * | 2019-03-13 | 2019-05-21 | 中国农业科学院植物保护研究所 | Metarhizium anisopliae IPPMHBC-009 composite bacteria agent and its application |
CN109769860A (en) * | 2019-03-12 | 2019-05-21 | 中国农业科学院植物保护研究所 | Metarhizium anisopliae IPPMHBC-009 composite bacteria agent and its application |
CN110876387A (en) * | 2019-12-05 | 2020-03-13 | 中国农业科学院植物保护研究所 | Compound bait for preventing and controlling locust and application thereof |
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 |
CN112970783A (en) * | 2021-03-02 | 2021-06-18 | 中国农业科学院植物保护研究所 | Composite bait agent of artemisia sieversiana crude extract and metarhizium anisopliae for preventing and treating locusts asiaticus and application of composite bait agent |
CN114437936A (en) * | 2021-12-02 | 2022-05-06 | 中国农业科学院植物保护研究所 | Metarhizium anisopliae IPPMHB614 and application thereof |
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
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CN109769860A (en) * | 2019-03-12 | 2019-05-21 | 中国农业科学院植物保护研究所 | Metarhizium anisopliae IPPMHBC-009 composite bacteria agent and its application |
CN109769861A (en) * | 2019-03-13 | 2019-05-21 | 中国农业科学院植物保护研究所 | Metarhizium anisopliae IPPMHBC-009 composite bacteria agent and its application |
CN110876387A (en) * | 2019-12-05 | 2020-03-13 | 中国农业科学院植物保护研究所 | Compound bait for preventing and controlling locust and application thereof |
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 |
CN112970783A (en) * | 2021-03-02 | 2021-06-18 | 中国农业科学院植物保护研究所 | Composite bait agent of artemisia sieversiana crude extract and metarhizium anisopliae for preventing and treating locusts asiaticus and application of composite bait agent |
CN114437936A (en) * | 2021-12-02 | 2022-05-06 | 中国农业科学院植物保护研究所 | Metarhizium anisopliae IPPMHB614 and application thereof |
CN114437936B (en) * | 2021-12-02 | 2023-06-20 | 中国农业科学院植物保护研究所 | Metarhizium anisopliae IPPMHB614 and application thereof |
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