CN110669675B - Metarhizium anisopliae MANGS71814 and application thereof in control of potato tuber moth - Google Patents
Metarhizium anisopliae MANGS71814 and application thereof in control of potato tuber moth Download PDFInfo
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- CN110669675B CN110669675B CN201911022041.XA CN201911022041A CN110669675B CN 110669675 B CN110669675 B CN 110669675B CN 201911022041 A CN201911022041 A CN 201911022041A CN 110669675 B CN110669675 B CN 110669675B
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- 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 provides a metarhizium anisopliae MANGS71814 and application thereof in prevention and control of potato tuber moth, the metarhizium anisopliae MANGS71814 is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms in 2019, 08 and 21 months, the preservation number is CGMCC No.18121, and the preservation address is No. 3 of Xilu No.1 of Beijing city morning-Yang district. The obtaining process of the metarhizium anisopliae MANGS71814 comprises the following steps: separating wild metarhizium anisopliae strain from collected morbid tuber moth 3-instar larva, culturing on Saccharum culture medium, transferring conidium to new culture medium, purifying, and performing single spore separation to obtain purified metarhizium anisopliae MANGS 71814. The metarhizium anisopliae MANGS71814 provided by the invention is a biocontrol fungus which can be used for controlling tuber moths, has the characteristics of strong pathogenicity to the tuber moths, environmental protection, no pollution and difficult generation of drug resistance, and can be widely used for controlling eggs, larvae and pupae of the tuber moths.
Description
Technical Field
The application belongs to the technical field of microorganisms, and particularly relates to metarhizium anisopliae MANGS71814 and application thereof in control of tuber moths of potatoes.
Background
The tuber moth (Phthyrmaea operculella (Zeller)) belongs to Lepidoptera, Mericidae and Mericidae, is a solanaceous plant pest mainly distributed in tropical zone and subtropical zone, and especially has serious harm to potatoes. In recent years, with global climate change, the distribution range of the insect is gradually spread, and the damage to solanaceous cash crops such as tomatoes and tobaccos is increased year by year.
In the prior art, chemical control methods are mainly adopted for controlling the tuber moths of potatoes at present, and the tuber moths have stronger drug resistance to a plurality of pesticides. In addition, the problems of pesticide residue and the like caused by a chemical prevention and control mode exist in the risks of potential safety hazards of people and animals and ecological environment influence.
Disclosure of Invention
In view of the above-mentioned defects of the prior art, the present application provides metarhizium anisopliae MANGS71814 and an application thereof in tuber moth control of potatoes.
The Metarhizium anisopliae (Metarhizium anisopliae) MANGS71814 provided by the invention is preserved in the China general microbiological culture Collection center on 21 days 08 and 9 months in 2019, the preservation number is CGMCC No.18121, and the preservation address is No. 3 of No.1 Hospital No.1 of West Chen of the sunward area in Beijing City.
The obtaining process of the metarhizium anisopliae MANGS71814 comprises the following steps: separating wild metarhizium anisopliae strain from collected morbid tuber moth 3-instar larva, culturing on Saccharum culture medium, transferring conidium to new culture medium, purifying, and performing single spore separation to obtain purified metarhizium anisopliae MANGS 71814.
The metarhizium anisopliae MANGS71814 is applied to the control of tuber moth (Phthoroma operculella (Zeller));
placing the eggs of potato tuber moth at a concentration of 2.0 × 103spore/mL-2.0X 108Soaking spores/mL metarhizium anisopliae MANGS71814 spore suspension for 5 s;
placing the 3 rd larva of potato tuber moth at the concentration of 2.0 × 103spore/mL-2.0X 108Soaking spores/mL metarhizium anisopliae MANGS71814 spore suspension for 5 s;
placing pupa of potato stem moth at concentration of 2.0 × 103spore/mL-2.0X 108spores/mL of Metarhizium anisopliae MANGS71814 spore suspension were immersed for 5 s.
The metarhizium anisopliae MANGS71814 and the application thereof in the control of the potato tuber moth have the following advantages:
1. the metarhizium anisopliae MANGS71814 strain obtained by separation is a fungus strain with strong pathogenicity on eggs, larvae and pupae of potato tuber moth.
2. The metarhizium anisopliae MANGS71814 strain is simple to culture, rapid in growth, large in spore yield, high in spore germination rate and easy to culture and propagate in a large scale indoors.
3. The conidia of the metarhizium anisopliae MANGS71814 strain has strong pathogenicity on the eggs, the larvae and the pupae of the tuber moth of the potato, is environment-friendly and pollution-free, is not easy to generate drug resistance, and can be widely used for preventing and treating the eggs, the larvae and the pupae of the tuber moth of the potato.
Detailed Description
Embodiments of the present application will be described in detail with reference to the following embodiments, so that how to implement the technical means for solving the technical problems and achieving the technical effects of the present application can be fully understood and implemented.
Entomopathogenic fungi are a class of fungi that infect insects through the surface of the insect and multiply on and in the body, resulting in the death of the insect. Because of being environment-friendly, has continuous control capability on pests and is not easy to generate drug resistance, the pesticide is considered to be the next generation of novel biological pesticide most likely to replace chemical pesticide.
Metarhizium anisopliae (Metarhizium anisopliae) belongs to the genus Metarhizium of the class Hyphomycetes. The inventor collects the metarhizium anisopliae MANGS71814 strain from the pupa body of the lepidoptera pest suffering from the disease in 8 months and 8 days in 2019 in Nannan, Xishuangbanna autonomous state, MengYunyuan town. After researching and collecting pupa bodies of lepidoptera insects which are naturally suffered from diseases in fields, a subject group to which the inventor belongs finds that the strain of metarhizium anisopliae MANGS71814 which has strong popularity and infection pathogenicity in fields has higher infection pathogenicity to potato tuber moth larvae than the metarhizium anisopliae KMa0107 strain which is separated from the larvae of the brown gill chafer suffering from diseases and is found before the subject group.
The following are examples of metarhizium anisopliae MANGS71814 provided by the invention and application thereof in control of tuber moth of potato.
Example I separation and identification of Metarhizium anisopliae MANGS71814
1.1 materials
1.1.1 test strains
The strain is collected from the pupa of Lepidoptera pest suffering from diseases in Nangong mountain of Mengyuan city, Yunnan province in 2019, 8 months and 8 days.
1.1.2 isolation and culture of the Strain
Collected 3-instar larvae of potato tuber moth infected with disease were returned to the laboratory in 5mL sterile EP tubes and were cultured by streaking in a light incubator (25. + -. 1) ℃ C., L// D16 h//8h, humidity 80% on Sasa (SDAY) medium (40 g glucose, 10g peptone, 10g yeast extract, 20g agar, pH natural). After culturing for 7 days, the conidia were transferred to a new medium for purification. The purified strain was stored in a 4 ℃ freezer using SDAY slant medium.
1.1.3 colony morphology Observation
The isolated and purified fungi were inoculated to SDAY medium (glucose 40g, peptone 10g, yeast extract 10g, agar 20g, pH natural), cultured in a light incubator at (25 + -1) ° C, L// D16 h//8h, and the colony diameter was measured and the colony morphology was observed at 3, 7 and 14D, respectively.
1.2 results
1.2.1 morphological identification results
After obtaining the pure culture metarhizium anisopliae MANGS71814 strain, the morphological characteristics of the obtained strain are identified. On SDAY medium, the colony is villous to flocculent, initially white, and olive green when producing spores. The separated branched hyphae can be seen under an optical microscope at 40 times, and are transparent and have the diameter of 1.5-2.0 mu m. The diameter of conidium peduncle is about 2 μm, the small bottle-shaped peduncle is generated at the end, and the size is 8-16 (-25) × 1.5-2 (2.2) μm. Conidia are produced in a basal manner from the end of the phialides, often interconnected to form long chains. The conidia are oblong to cylindrical, the two ends are blunt and truncated to blunt circles, and the size change range is 5-7 (-9) × 2-3 (4.5) mum. When the spores exist singly, the color is bright, when the spores exist in a pile, the spores are olive green, and the fallen conidia are often gathered into rhombic spore lumps.
EXAMPLE two determination of the pathogenicity of Metarhizium anisopliae MANGS71814 to eggs, larvae and pupae of potato tuber moth
1 Material
1.1.1 test insects
The tuber moths were collected from the potato fields of Xuanwei, Yunnan, and were continuously raised indoors for 5 generations, and the experiments were conducted in an intelligent artificial incubator (RG-300) at a temperature of 25 + -1 deg.C, a Relative Humidity (RH) of 60 + -5%, and a photoperiod L: D of 16: 8. The tuber moth of potato is raised by using fresh potato blocks and leaves.
1.1 spore suspension preparation
Inoculating the separated strain to SDAY culture medium, scraping conidia on the surface of the culture medium by using a sterile scalpel after the strain produces a large amount of conidia (about 20d), placing the conidia in 0.02% sterile Tween-80, and preparing into 2 × 103–2×108spores/mL of spore suspension.
1.1.2 impregnation method
Selecting healthy tuber moth 3-instar larvae with consistent sizes, inoculating by adopting a dipping method, putting the tuber moth larvae into the spore suspension for dipping for 5s, taking out, and then sucking redundant water on the surfaces of the larvae by using sterilized filter paper. Then, they were placed in 12-well cell culture plates with added potato leaves and were kept in a 16 h/8 h light incubator at 25 ℃. Observing regularly every day, preserving moisture and culturing dead insects under the conditions of (25 +/-1) DEG C and 16 h/8 h/L// D, and determining the death reason of each insect according to whether the insect grows out target bacteria. 3 replicates were set, 20 beetles per replicate.
1.1.3 data analysis
Excel 2010 is adopted for data arrangement, and a toxicity regression equation is calculated by means of machine value analysis of DPS data processing system 14.0 software.
2. Results
2.1 infection lethality of Metarhizium anisopliae MANGS71814 to potato tuber moth eggs
As can be seen from Table 1, the ratio was 2.0X 103–2.0×108At the spore concentration per mL, metarhizium anisopliae MANGS71814 shows infection pathogenicity on potato tuber moth eggs, and the infection fatality rate is increased along with the increase of the inoculation depth. At 2.0X 106、2.0×107、2.0×108At spore concentration per mL, mortality rates of potato tuber eggs reached 83.33%, 96.67% and 100% at day 3 post-inoculation. Therefore, the metarhizium anisopliae MANGS71814 has strong pathogenicity on the eggs of the potato tuber moth.
TABLE 1 lethality (%) of Metarhizium anisopliae MANGS71814 to potato tuber moth eggs
2.2 infection lethality of Metarhizium anisopliae MANGS71814 against Botrytis cinerea larvae
As is clear from Table 2, the ratio is 2.0X 103–2.0×108Inoculating 2.0 × 10 of metarhizium anisopliae MANGS71814 to the 3 rd day of 3 rd larva of potato tuber moth when the larva is infected by the metarhizium anisopliae in the spore concentration per mL3、2.0×104、2.0×105、2.0×106、2.0×107And 2.0X 108After inoculation at spore concentration per mL, the mortality rates of 3 rd larvae of potato tuber moth are respectively (83.33 +/-0.60)%, (90.00 +/-0.30)%, (93.33 +/-0.68)%, (98.33 +/-0.47)%, 100% and 100%. Therefore, the metarhizium anisopliae MANGS71814 has strong pathogenicity of infection to the larvae of the potato tuber moth.
TABLE 2 lethality (%) of Metarhizium anisopliae MANGS71814 against potato tuber moth larvae
2.3 infection lethality of Metarhizium anisopliae MANGS71814 to potato tuber moth pupae
As can be seen from the results in Table 3, the value is 2.0X 103–2.0×108The cumulative mortality rate of the metarhizium anisopliae MANGS71814 infected potato tuber moth pupa on the 1 st day under the spore concentration per mL is respectively (15.00 +/-0.34)%, (28.33 +/-1.16)%, (51.66 +/-1.35)%, (85.00 +/-0.77)%, 100% and 100%. I.e. at 2.0 x 107And 2.0X 108After inoculation of the spore concentration per mL, the infection lethality rate of the potato stem moth pupae on 3 days reaches 100%. Therefore, the metarhizium anisopliae MANGS71814 has strong pathogenicity of infection to the larvae of the potato tuber moth.
TABLE 3 mortality of Metarhizium anisopliae MANGS71814 to Bombycis potamota pupae (%)
In conclusion, the metarhizium anisopliae MANGS71814 provided by the invention is a biocontrol fungus which can be used for controlling tuber moths, has the characteristics of strong pathogenicity to the tuber moths, environmental protection, no pollution and difficulty in generating drug resistance, and can be widely used for controlling eggs, larvae and pupae of the tuber moths.
The foregoing description shows and describes several preferred embodiments of the present application, but as aforementioned, it is to be understood that the present application is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of numerous other combinations, modifications, and variations within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the application, which is to be protected by the claims appended hereto.
Claims (1)
1. Metarhizium anisopliae (Metarhizium anisopliae)Metarhizium anisopliae) MANGS71814 which is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms (CGMCC) at 21.08.2019, with the preservation number of CGMCC number 18121 and the preservation address of No. 3 Siro No.1 of Beijing, Chaoyang, the quarter;
the obtaining process of the metarhizium anisopliae MANGS71814 comprises the following steps: separating wild metarhizium anisopliae strains from collected morbid tuber moth 3-instar larvae, culturing on a Sachs culture medium, transferring conidia to a new culture medium for purification, and then performing single spore separation to obtain purified metarhizium anisopliae MANGS 71814;
the metarhizium anisopliae MANGS71814 is arranged on the tuber moth of potatoPhthorimaea operculella（Zeller）]The application in the aspect of prevention and control:
placing the eggs of potato tuber moth at a concentration of 2.0 × 107spore/mL-2.0X 108Soaking spores/mL metarhizium anisopliae MANGS71814 spore suspension for 5 s;
placing the 3 rd larva of potato tuber moth at the concentration of 2.0 × 106spore/mL-2.0X 108Soaking spores/mL metarhizium anisopliae MANGS71814 spore suspension for 5 s;
placing pupa of potato stem moth at concentration of 2.0 × 107spore/mL-2.0X 108spores/mL of Metarhizium anisopliae MANGS71814 spore suspension were immersed for 5 s.
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| CN111662828B (en) * | 2020-06-30 | 2022-04-12 | 福建省农业科学院植物保护研究所 | Metarhizium leylanicum and application thereof |
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| CN104611240A (en) * | 2015-02-16 | 2015-05-13 | 重庆聚立信生物工程有限公司 | Application of Metarhizium anisopliae strain in preparation of insecticide for vegetable pest insects |
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| CN104611240A (en) * | 2015-02-16 | 2015-05-13 | 重庆聚立信生物工程有限公司 | Application of Metarhizium anisopliae strain in preparation of insecticide for vegetable pest insects |
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| CN109055236A (en) * | 2018-08-20 | 2018-12-21 | 云南农业大学 | A kind of thin foot Isaria WSWM1171 and its application in the prevention and treatment of potato tubers chrysalis |
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