CN113293142B - Boston crinis nuclear polyhedrosis virus strain as well as propagation method and application thereof - Google Patents

Boston crinis nuclear polyhedrosis virus strain as well as propagation method and application thereof Download PDF

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CN113293142B
CN113293142B CN202110258353.1A CN202110258353A CN113293142B CN 113293142 B CN113293142 B CN 113293142B CN 202110258353 A CN202110258353 A CN 202110258353A CN 113293142 B CN113293142 B CN 113293142B
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占智高
靳亮
况文东
关丽梅
王金昌
陈俊晖
刘卓荣
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INSTITUTE OF MICROBIOLOGY JIANGXI ACADEMY OF SCIENCES
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Abstract

The invention discloses a mountain crawling tiger hornworm nuclear polyhedrosis virus strain and a propagation method and application thereof, wherein the strain is the mountain crawling tiger hornworm nuclear polyhedrosis virus; the preservation name is the Nanchang strain ThjaNPV-NC of the Cladosporus reyngii nuclear polyhedrosis virus. The invention improves the yield and safety of green fruits and vegetables, reduces the pollution of chemical pesticides to the environment and protects the ecological environment.

Description

Boston crinis nuclear polyhedrosis virus strain as well as propagation method and application thereof
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a mountain tiger hornworm nuclear polyhedrosis virus strain, a propagation method and application thereof.
Background
Parthenocissus tricuspidata is a different name for various plants. Groveling partridge, red vine, barnacle, dragons, maple vine, small worm croaker, red sage, red kudzu, and Vitaceae plant. The fruits of the parthenocissus tricuspidata can be used for brewing wine, and the roots, the stems and the leaves can be used as medicines for breaking blood stasis and eliminating swelling and toxin. Jiangxi is also a Chinese herbal medicine province, and the prevention and treatment of the Chinese herbal medicine of the partridge asiatic follows a method of environmental protection, less pollution and less residue.
Grape (vitas vinifera L.) is a woody vine plant of the genus Vitis of the family Vitaceae, is one of the oldest fruit tree species in the world, is frequently planted in places such as Meiling of Nanchang city, jiangxi province, and the like, is a famous fruit, is eaten raw or dried, and is brewed, wine lees after brewing can be used for increasing wine food acidity, and roots and vines can be used for preventing vomiting and miscarriage. The living standard of people is improved, the problem of pesticide residue on fruits and vegetables is particularly concerned, the novel microbial pesticide is found for scientifically and effectively preventing and treating the pests of the fruits and the vegetables to replace the chemical pesticide, the ecological environment is stabilized, and the biological pesticide is a major scientific problem of pest prevention and control research.
The Gekko Swinhonis is also called grape prodenia litura and sparrow leaf moth (Thertara jpoinca), and belongs to Lepidoptera and Gekkonidae. The main parasitic plants: grapes, taros, tigers, ailanthus altissima, camphor trees, black cypress and the like almost all over provinces in the country, generally, 6-10 months of damage is continued, when the damage is particularly serious, leaves of plants are almost completely removed, leaves, branches and the like are only left, even the whole plants die, and the growth and the application of the plants are seriously influenced. The adoption of such pesticides as dichlorvos emulsifiable solution, high-efficiency cypermethrin, acetamiprid, phoxim and the like at home and abroad has the disadvantages of high cost, insecurity to aquatic animals and human beings and the like, and the overproof pesticides can cause fruits and vegetables to lose edible value and cause great economic loss to farmers, so that the pure biological pesticides are gradually popularized, and the development and selection of new novel green high-efficiency biological pharmaceutical varieties are particularly important for effectively controlling pests.
The insect accords with the development trend of transforming the pesticide from the traditional organic chemical substance to the 'environment-friendly pesticide' or the 'biological reasonable pesticide', and is favored by researchers and users at home and abroad.
The insect Nuclear Polyhedrosis Virus (NPV) belongs to the baculovirus family, mainly takes agricultural and forestry pests of Lepidoptera, diptera, hymenoptera and the like as hosts, has the characteristics of pure nature, environmental protection, strong specificity, no environmental pollution, no harm to human and livestock and the like, and can be developed into safe and effective biological insecticides.
Disclosure of Invention
Aiming at the defects of the prior art, the first purpose of the invention is to provide a nuclear polyhedrosis virus strain of a new mountain crawling moth and the application thereof in the aspect of preventing and controlling important pests, improve the yield and safety of green fruits and vegetables, reduce the pollution of chemical pesticides to the environment and protect the ecological environment.
It is a second object of the invention to provide the viral characteristics of the invention.
A third object of the present invention is to provide the use of the present invention for controlling important pests.
The invention is realized by the following technical scheme.
A strain of the mountain climbing tiger hornworm nuclear polyhedrosis virus is preserved in China center for type culture collection, the preservation address is China center for type culture collection, wuhan university, wuhan, the preservation date is 2020, 9 months and 9 days, the preservation name is mountain climbing tiger hornworm nuclear polyhedrosis virus Nanchang strain ThjanPV-NC, the preservation number is CCTCC NO: v202058.
A propagation method of a Botrytis pernyi nuclear polyhedrosis virus comprises the following steps:
selecting three-or four-instar cutworm larvae as proliferation host, and selecting 1 × 10 larvae as proliferation host 5 Coating fresh cayratia japonica leaves on PIB/mL polyhedrosis suspension of the cutworm nucleopolyhedrovirus, and feeding in an incubator at 27 ℃ and 50-70% relative humidity;
after two to three days of poison feeding, the larvae begin to die;
collecting dead larvae every day until all larvae die;
after the larva infects the nuclear polyhedrosis virus of the tiger creeper, the polypide is liquefied, milk white liquid containing a large amount of virus polyhedrosis particles flows out, the milk white solution is centrifuged at 800rpm for 3-5min, and insect tissue residues are taken out to obtain a large amount of milk white virus crude extract containing the propagated nuclear polyhedrosis virus of the tiger creeper.
The invention relates to an application of a nucleopolyhedrosis virus strain of a tiger moth in preparation of a biological insecticide.
Compared with the prior art, the invention has the advantages that: the invention improves the yield and safety of green fruits and vegetables, reduces the pollution of chemical pesticides to the environment and protects the ecological environment.
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FIG. 1 is a wild-collected, diseased and dead phenotype of cutworm hornworm according to the present invention;
FIG. 2 is a scanning and transmission electron microscope image of the athyria reptans nuclear polyhedrosis virus ThjanPV-NC of the invention; a in FIG. 2 is a scanning electron microscope image; scale bar:2 mu m; b in FIG. 2 is a transmission electron micrograph; scale bar: scale bar 200nm;
FIG. 3 is an electrophoretogram of the DNA of the nucleopolyhedrosis virus genome of Gekko Swinhonis of the present invention (M: takara DL5000;1: gekko Swinhonis nucleopolyhedrosis DNA);
Detailed Description
The invention is further illustrated by the following examples. It should be understood that these examples are illustrative and exemplary of the present invention, and are not intended to limit the scope of the present invention in any way.
Example 1: separation and purification of mountain creeper hornworm nuclear polyhedrosis virus
The virus of the invention is separated from a sample of a naturally dead cutworm hornworm cadaver in a high and new area Ai Xihu wetland park Cayratia japonica (scientific name: cayratia japonica (thunb.) Gagnep.) in Nanchang city, jiangxi, and the corpse is inversely hung on branches when found (figure 1). After the sample is collected, the sample is put into a 15mL corning tube and brought back to the experimental identification. The next day, the cadaver sample is found, the epidermis is cracked, a large amount of white milky white liquid flows out, and the typical phenotype of the death of the nuclear polyhedrosis virus appears, so that the pathogenic microorganism is judged to be the nuclear polyhedrosis virus, and the virus is named as the Nanchang strain of the nuclear polyhedrosis virus of the cutworm moth.
The resulting milky white liquid was centrifuged at 800rpm for 3-5min to remove the insect tissue residue, and the milky white liquid was pipetted into a new 15mL tube. Centrifuging at 3000-5000rpm for 20-40min to precipitate virus polyhedron particle. Adding water suspension polyhedron particles, repeating the above two centrifugation steps for 2-3 times, washing polyhedron particles to obtain relatively pure virus polyhedron precipitate, and suspending with water to obtain virus solution.
Example 2: electron microscope observation of Spodoptera litura nuclear polyhedrosis virus
Samples were prepared from viral polyhedra using the method described by Zhan et al (Zhan et al, ji, 2019), and it was observed by electron microscopy that each polyhedron particle contained a number of virus particles varying in number, and the polyhedron particle appeared polygonal with a particle size of 1.41 ± 0.15 μm (N = 18) (Average ± SEM) (fig. 2). Further, since the sphingan operculella nuclear polyhedrosis virus is a single-particle-embedded type, the name of the virus is named as the name of the novel japonica single nuclear polyhedrosis virus isolate Nanchang, abbreviated in english, according to the naming rules: thjanPV-NC.
Example 3: production method or propagation method of Spodoptera litura nuclear polyhedrosis virus
In order to obtain a large amount of nucleopolyhedrosis virus of cutworm hornworm, the larva of cutworm hornworm of three-instar or four-instar is taken as a proliferation host, and 1 × 10 5 Coating fresh cayratia japonica leaves on PIB/mL polyhedrosis suspension of the cutworm nucleopolyhedrovirus, and feeding in an incubator at 27 ℃ and 50-70% relative humidity; after two to three days of feeding poison, the larvae begin to die; dead larvae were collected daily until all larvae were dead. After the larva infects the tiger nuclear polyhedrosis virus, the polypide is liquefied, milky white liquid containing a large amount of virus polyhedrosis particles flows out, and the virus polyhedrosis particles can be completely released only by slightly grinding with a gun head or a mortar; centrifuging the milky white solution at 800rpm for 3-5min to obtain insect tissue residue, and collecting milky white coarse extractive solution containing proliferated nucleopolyhedrosis virus of Gekko Swinhonis.
Example 4: extracting and electrophoretic analysis of mountain tiger hornworm nuclear polyhedrosis virus genome
Taking 200 μ L of the extract containing 1 × 10 11 Adding 100 mu L of 3-fold DAS buffer solution into virus solution of polyhedron, incubating in water bath at 37 ℃ for 1-5h, splitting the nucleopolyhedrosis virus particles of the athyris reptans, adding SDS with final concentration of 0.5% and proteinase K with concentration of 50mg/mL, incubating overnight at 37 ℃ for digestion, extracting with phenol, phenol-chloroform mixed solution and chloroform sequentially, precipitating the genomic DNA with absolute ethyl alcohol, dissolving the precipitate with 100 mu L of water to obtain genomic DNA solution, taking 2 mu L of the genomic DNA solution, and analyzing by 0.5% agarose gel electrophoresis, the result is shown in figure 3.
Example 5: sequencing conserved gene lef-9, lef-8, polh of nuclear polyhedrosis virus of tiger
The primers shown in Table 1 were used to amplify the fragments lef-9, lef-8 and polh, respectively, the PCR products were electrophoresed through 1% agarose gel, the fragments were recovered by gel cutting, and the fragments were sequenced by Shanghai Biotech (Shanghai) to obtain the sequences of the 3 conserved genes.
TABLE 1 primers and primer sequences used
Primer name Sequence
prPH-1 TGTAAAACGACGGCCAGTNRCNGARGAYCCNTT
prPH-2 CAGGAAACAGCTATGACCDGGNGCRAAYTCYTT
prL8-1 CAGGAAACAGCTATGACCCAYGGHGARATGAC
prL8-2 CAGGAAACAGCTATGACCAYRTAS1GGRTCYTCS2GC
prL9-1 CAGGAAACAGCTATGACCAARAAYGGITAYGCB3G
prL9-2 TGTAAAACGACGGCCAGTTTG4TCDCCRTCRCARTC
The sequence results are shown below:
>lef-9Theretra japonica nucleopolyhedrovirus isolate Nanchang(ThjaNPV-NC),partial cds
GGCCAATTTTTGTACAGCACATTCCTTAACACTGCGAGTTTTTACTCAAACGTGCAGTGCATAAACGGCACCAATGAAATTGAGCCGCCGAAAGCCAGTATTAGACGGTACTACGGTCGTGACGTGCACGACATACGCGCCTGGACCACGCGCCATCCGTGTATATCGCAACTGAACACACAGATTGCCAAAGTGCGCCAACCTGAAGATTACTTAAACTGGAACGCCAAGATAGGCTTGGGAACATGTAATGGCGCTCATCGCGACT
>lef-8 Theretra japonica nucleopolyhedrovirus isolate Nanchang(ThjaNPV-NC),partial cds
ATGACAGTGGCGGGTAAGAAATTTTTTCTGTGTCGCAACGTACGTCTGCCAAGTGTAGACTATCAATTGGTCAGCGATAAATTTGCAAAACTCGAATCCGACAATCTTATTTTTCGAAACGACCATAACACTAACGACACGGAACGTTTGATTGTAGCGTTTAACGATAGACCAACAATATTTGCTTGTAACCGTAAAGACTTGTTTAAAATATTTTATGAGTTAAAACGAGAAATGTGTCCGATTGAATTAAAGCTAAGCGACGGCATTCTGTTTGTCAATCATCACGAAGGTATGGTTTGCCTAAAACGCACTGTTCAAATACATGACGATGCCAAAATACAAACATTGCTCACGTCGTACGAGTATCACAACAAATCGTCGTTGCTCAAGACCACATCGGATGTGGGAACAATACACGAAAACGACAACGTTACTAGTTTGATGTCTAAGCTAGTCCAATACTATTACAAAGGGTACACAAGAATATTTAGCACAATACCAGTGCCCAAGATGATTGTGTCTTTGACGAATTTGAAAAACGCCATGCCCATACTTAAATGCGAAGGCGAAAATTTAAATCGAACAAAATTTTTGGACAATTTACCGATGGGCAATTCAGTGTTGGTGTCTCCTCACATACAAGTCAACAACAAAATGTTCAAATTGTGGACTTTGGTGAGGGATAATCGACTTTTAACGGCCGAAGA
>polh Theretra japonica nucleopolyhedrovirus isolate Nanchang(ThjaNPV-NC),partial cds
TTTTTTAGGGCCTGGAAAAAACCAAAAACTAACATTATTTAAAGAAATTAGAAGCGTTAAACCTGATACTATGAAACTAGTCGTAAACTGGAGCGGTAAAGAGTTTTTGAGAGAAACTTGGACACGTTTTATGGAGGACAGCTTTCCTATCGTTAATGACCAAGAAATCATGGACGTTTTCCTAGTCATCAATCTCAGACCAACTAAACCCAACAGGTGTTATAGGTTTTTGGCACAACACGCTCTGCGTTGTGATCCTGACTATGTTCCTCACGAAGTCATTCGCATTGTGGAGCCTTCGTATGTGGGTAGCCACAACGAGTACCGTATCAGTCTTGCCAAACGAGGAGGAGGCTGTCCCGTCATGAACCTGCATTCTGAATATACTAATTCCTTCGAAGAGTTTATTCACAAAGTGATCTGGGAGAACTTCTACAAACCAATTGTGTACATTGGCACAGATTCAGCTGAGGAAGAAGAAATCCTGATGGAAGTTTCGTTAGTGTTCAAGATTAAAGAATT
example 6: determination of insecticidal capacity of Helicoverpa reptans nuclear polyhedrosis virus
Diluting the nucleopolyhedrosis virus of Gekko Swinhonis with water to 1 × 10 8 、1×10 7 、1×10 6 、1×10 5 And 1X 10 4 5 concentrations of PIB/mL, using sterile water as a control, spraying the fresh leaves with the same volume of virus solution or water, after the water on the leaves is dried, inoculating 30 heads of larvae of the cutworm hornworm with the age of 3 in each insect-raising box, and repeating the treatment for 3 times. The animals were kept in an incubator at 27. + -. 1 ℃ and 60% relative humidity, and the number of dead and live insects was checked and recorded day by day, and the corrected mortality was calculated, as shown in Table 2. The insecticidal capacity data shows that the virus has strong insecticidal capacity on the cutworm hornworm and has good development potential.
Table 2: prevention and treatment effect of nucleopolyhedrovirus of cutworm on 3 larvae of cutworm
Concentration of virus (PIB/mL) Corrected mortality%
1×10 4 62
1×10 5 83
1×10 6 92
1×10 7 100
1×10 8 100
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the spirit and scope of the invention.
Figure IDA0003904057480000011
Figure IDA0003904057480000021

Claims (3)

1. A strain of Botrytis pervirens nuclear polyhedrosis virus is characterized in that: the strain is preserved in a China center for type culture Collection, the preservation address is China center for type culture Collection of Wuhan university, wuhan, the China city, the preservation date is 2020, 9 and 10 days, the preservation name is the mountain climbing tiger hornworm nuclear polyhedrosis virus Nanchang strain ThjanPV-NC, the preservation number is CCTCC NO: v202058.
2. The method for propagating the athea pernyi nuclear polyhedrosis virus of claim 1, comprising the steps of:
selecting three-or four-instar cutworm larvae as proliferation host, and selecting 1 × 10 larvae as proliferation host 5 Coating fresh cayratia japonica leaves on PIB/mL polyhedrosis suspension of the cutworm nucleopolyhedrovirus, and feeding in an incubator at 27 ℃ and 50-70% relative humidity;
after two to three days of feeding poison, the larvae begin to die;
collecting dead larvae every day until all larvae die;
after the larva infects the nuclear polyhedrosis virus of the tiger creeper, the polypide is liquefied, milk white liquid containing a large amount of virus polyhedrosis particles flows out, the milk white solution is centrifuged at 800rpm for 3-5min, and insect tissue residues are taken out to obtain a large amount of milk white virus crude extract containing the propagated nuclear polyhedrosis virus of the tiger creeper.
3. The use of the nucleopolyhedrosis virus strain of cutworm webworm as defined in claim 1 in the preparation of an insecticide for killing cutworm webworm.
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