CN112342199A - Spodoptera frugiperda virus-resistant pesticide production strain and preparation method and application thereof - Google Patents
Spodoptera frugiperda virus-resistant pesticide production strain and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/40—Viruses, e.g. bacteriophages
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
- C12N2710/14151—Methods of production or purification of viral material
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Abstract
The invention discloses a spodoptera frugiperda virus resistant pesticide production strain, a preparation method and application thereof, and relates to the technical field of biological control of agricultural pests. The Spodoptera frugiperda virus resistant insecticide production strain is obtained by cloning a wild Spodoptera frugiperda nuclear polyhedrosis virus Hub through a polypide, and the virus strain has the same insecticidal activity as the wild virus, but has higher virus yield, and provides a reliable production strain for researching Spodoptera frugiperda virus insecticides.
Description
Technical Field
The invention belongs to the technical field of biological control of agricultural pests, and particularly relates to a spodoptera frugiperda virus resistant pesticide production strain, and a preparation method and application thereof.
Background
The spodoptera frugiperda invades China for the first time in 2019, namely the insect-catching area in China reaches more than 1500 ten thousand mu, and the actual damage area is 246 ten thousand mu. Spodoptera frugiperda has been colonized in south China as migratory pests, and meanwhile, foreign insect sources continuously migrate, so that the situation is severe in 2020 and years later, and the prevention and control tasks are harder. The biological insecticide taking the insect virus as the insecticidal active ingredient has the advantages of high insecticidal activity, difficult generation of resistance by pests, capability of causing epidemic diseases of pest populations and the like, and is an effective means for preventing and controlling invasive pests.
Baculoviruses are an important group of pathogens that can infect a variety of pests. The field observation shows that Spodoptera frugiperda is susceptible to Spodoptera frugiperda nuclear polyhedrosis virus (SfMNPV), which can cause natural epidemic disease, so that SfMNPV can be used as a biological insecticide. Although several SfMNPV isolates from usa, nigla melon, brazil, argentina, columbia and mexico have been biologically and/or genetically identified, it is still necessary to select for the development of isolates suitable for the control of local spodoptera frugiperda and to characterize the isolates obtained in different geographical regions. Some studies have shown that local isolates of Spodoptera frugiperda nucleopolyhedrovirus have greater potential for controlling local Spodoptera frugiperda (S) ((S))
Garcia et al, 2019.Molecular and Molecular characterization of multiple nuclear polyhedrogens from Mexico and the inductive activity of the induced Spodoptera frugiperda (Lepidotera: Noctuidae) Journal of Applied biology 144: 123-.
Therefore, a Spodoptera frugiperda virus insecticide production strain needs to be researched, has higher virus yield while meeting insecticidal activity, and is better applied to local control of Spodoptera frugiperda for popularization and application.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a Spodoptera frugiperda poison insecticide production strain and a preparation method thereof, which meet insecticidal activity and have higher virus yield.
In order to achieve the aim, the invention provides an insecticide production strain for resisting Spodoptera frugiperda virus, which is obtained by cloning Spodoptera frugiperda nuclear polyhedrosis virus Hub through polypide, wherein the Spodoptera frugiperda nuclear polyhedrosis virus Hub is preserved in China center for type culture collection (CCTCC NO: V202054) in 9.3.2020.
Another object of the present invention is to provide a method for preparing the above-mentioned spodoptera frugiperda virus-resistant insecticide-producing strain, comprising the steps of:
1. infecting Spodoptera frugiperda with Spodoptera frugiperda nuclear polyhedrosis virus Hub, collecting virus lethal larva separately, and purifying virus inclusion body;
2. repeating the step 1 for two times;
3. extracting the inclusion body virus DNA, separating and active screening to obtain the production strain.
Further, the infection was effected by a dropping method, and the Spodoptera frugiperda nucleopolyhedrovirus Hub concentration was a concentration causing 5% mortality.
Further, the spodoptera frugiperda is spodoptera frugiperda at the beginning of 4 years.
Further, the separation step is carried out with cleavage with PstI, and the cleaved fragments are separated by agarose gel electrophoresis.
Further, the activity screening step comprises the steps of infecting 2-year-old Spodoptera frugiperda by a liquid drop method until all the larvae die or pupate, calculating half lethal concentration LC50 of beet armyworm larvae by utilizing probit regression, and taking a virus type with low LC50 as a production strain.
The invention also aims to provide the application of the Spodoptera frugiperda nucleopolyhedrosis virus production strain in preventing and controlling Spodoptera frugiperda.
The preparation principle of the invention is as follows:
1) by utilizing the characteristic that wild insects carry insect viruses, a large number of insects are collected in the field and are intensively raised indoors to obtain natural insecticidal viruses;
2) different genotypes of baculovirus are obtained by utilizing a worm cloning technology, the principle of the worm cloning technology is that when host insect larvae are infected by low-dose virus (for example, concentration causing 5% of mortality, LC5), most of infection is caused by propagation of single virus, the virus contained in a single dead insect is collected and recorded as a clone, molecular biological analysis, such as restriction enzyme map analysis, the host insect is re-infected by the clone with the wild type virus map by using LC5 concentration, the larvae which are killed by the virus are re-collected as single insects, and the operation is carried out for 3 times in total, so that the virus with a single genotype can be separated and purified;
3) the genotype obtained by utilizing the insect body cloning technology is subjected to biological activity measurement and virus yield measurement to obtain the virus pesticide production strain with excellent performance.
Compared with the prior art, the invention has the beneficial results that:
1. the production strain obtained by separating the baculovirus genotype from the wild type virus by the method has higher virus yield while meeting the insecticidal activity;
2. provides an excellent production strain for the research and development and control of virus insecticide of the exotic invasive pest Spodoptera frugiperda.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 shows scanning electron micrographs (A) and ultrathin sections (B) of Spodoptera frugiperda nuclear polyhedrosis virus SfMNPV-Hub strain.
FIG. 2 PCR products of the Spodoptera frugiperda nuclear polyhedrosis virus SfMNPV-Hub strain polh, lef-8 and lef-9 genes.
FIG. 3 shows PstI restriction electrophoretograms of Spodoptera frugiperda nuclear polyhedrosis viruses SfMNPV-Hub, SfMNPV-Hub1 and SfMNPV-Hub 2.
FIG. 4 is a graph comparing the yields of monoworms for SfMNPV-Hub, SfMNPV-Hub1 and SfMNPV-Hub 2.
Detailed Description
The following examples are intended to illustrate the invention without limiting its scope. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.
Example 1
The method comprises the following steps of obtaining wild Spodoptera frugiperda nuclear polyhedrosis virus:
1) the preparation method of the spodoptera frugiperda artificial feed comprises the following steps of preparing 150g of corn flour, 87g of soybean flour, 30g of yeast powder, 15g of casein, 15g of agar powder, 1g of sorbic acid, 2g of nipagin, 1g of sodium benzoate, 0.2g of cholesterol, 0.1g of inositol, 0.5g of choline chloride, 0.1g of wecker salt, 0.5g of compound vitamin, 2.2 g of vitamin C, 2mL of rapeseed oil and 770mL of tap water per 1 liter of artificial feed.
2) Collecting Spodoptera frugiperda larvae from a corn field in Hubei province, bringing the Spodoptera frugiperda larvae back to a laboratory for breeding a large amount of Spodoptera frugiperda collected from the corn field indoors by using the artificial feed provided in the step 1), collecting larvae with typical virus infection symptoms (such as slow movement, swollen somite, thinned epidermis and the like), and carrying out virus separation and purification on dead larvae with milky white turbid liquid in vivo.
3) The virus was observed by scanning electron microscopy to be polyhedral in morphology and approximately 1.39. + -. 0.23 μm in size (FIG. 1A), and the section of the polyhedron was observed by transmission electron microscopy to find that the virus particles were rod-shaped, multi-particle-embedded, and approximately 38X 306nm in size (FIG. 1B).
4) A sample (1.0 g or 1.0 mL) was placed in a 100mL centrifuge tube and 99.0mL of distilled water was shaken on a shaker for 1 min. And (3) putting 300 mu L of the shaken suspension into a centrifuge tube, adding 100 mu L of 3 Xalkali lysate, and placing the centrifuge tube into a water bath at 37 ℃ for 30 min. Taking out, adding 200 mu L of Tris-HCl buffer solution, centrifuging for 8min at 10000 Xg, taking out supernatant, adding 5 mu L of proteinase K and 60 mu L of SDS, then placing in 65 ℃ water bath for 2h, taking out, cooling to room temperature, adding 650 mu L of Tris saturated phenol, uniformly mixing, centrifuging for 5min at 10000 Xg, taking out supernatant to a new centrifugal tube, adding 650 mu L of phenol: chloroform (1:1) was mixed well, centrifuged at 10000 Xg for 5min, the supernatant was taken out into a new centrifuge tube, and 650. mu.L of chloroform: isoamyl alcohol (24:1) is mixed evenly, centrifuged for 5min at 10000 Xg, the supernatant is taken out and put into a new centrifuge tube, the DNA concentration is measured by an ultraviolet spectrophotometer, and the mixture is put at 4 ℃ for standby.
5) 10-50ng of the extracted viral DNA was taken, and 2. mu.L (10mM) of each of Taq enzyme 1. mu.L, corresponding 10 XTaq enzyme buffer 3. mu. L, dNTP 2. mu. L, polh gene, lef-8 gene and upstream and downstream primers of lef-9 gene was added thereto, sterilized pure water was added thereto to 30. mu.L, and the mixture was placed in a PCR apparatus for PCR amplification. PCR amplification conditions of polh gene: after heat denaturation at 95 ℃ for 3min, 34 cycles of the following were carried out: 95 ℃ for 30s, 50 ℃ for 1min, 72 ℃ for 1min, and finally 72 ℃ for 5 min. The LEF-8 gene PCR amplification conditions are as follows: after thermal denaturation at 95 ℃ for 2min, 34 cycles of the following were carried out: 95 deg.C for 2min, 48 deg.C for 1min, 72 deg.C for 1min, and finally 72 deg.C for 5 min. The LEF-9 gene PCR amplification conditions are as follows: after heat denaturation at 95 ℃ for 3min, 34 cycles of the following were carried out: 30s at 95 ℃, 15s at 47 ℃, 30s at 72 ℃ and finally 5min at 72 ℃. And (3) taking 1 mu L or 1kb DNA Marker of the PCR amplification products of the polh gene, the lef-8 gene and the lef-9 gene, uniformly mixing the products with a DNA loading buffer solution, sequentially spotting the mixture in agarose gel, and performing electrophoresis at 60V until a bromophenol blue band reaches the other end of the gel. After electrophoresis, DNA staining was performed for 5min in ethidium bromide solution, and PCR products were observed in a gel imaging system. The PCR products of the polh, lef-8 and lef-9 genes were 0.5kb, 0.7kb and 0.27kb, respectively (as shown in FIG. 2, lanes 1, 2 and 3 are PCR products of the polh, lef-8 and lef-9 genes, respectively).
6) The PCR product was DNA sequenced with the m13 primer. The DNA sequence of the polh gene, the lef-8 gene and the lef-9 gene which are detected by DNAMAN software is compared with the DNA sequence of the corresponding gene of SpfrNPV (Colombian strain).
7) PCR amplification and sequencing was performed with universal primers for the baculovirus polh, lef-8 and lef-9 genes, with the following results (partial sequence):
partial sequence of Spodoptera frugiperda nucleopolyhedrovirus Hub polh gene:
GGCATTCTGCACGGCAAACCATAGCTGACTCTGTTCAAAGAGATCCGTATCGTCAAGCCCGACACCATGAAGCTGGAAGTAAACTGGAGCGGCAAAGAGTTTCTCAGGGAAACTTGGACCCGTTTCATGGAAGACAGCTTCCCTATCGTGAACGACCAAGAAATTATGGACGTGTTCCTAGTGATCAACATGAGACCCACTAGGCCCAACAGATGTTTCAGATTCCTGGCGCAACACGCTCTCCGTTGCGACCCTGACTACGTTCCTCACGAAGTCATTCGCATTGTGGAGCCCGTGTACGTAGGAAACAACAACGAATACCGCATCAGCCTGGCCAAGAAGGGCGGCGGCTGCCCTGTCATGAACCTTCACTCTGAGTACACGCACTCCTTCGAAGAGTTCATCAACCGTGTCATCTGGGAGAACTTCTACAAACCCATCGTCTACGTAGGAACCGACTCTGGTGAGGAAGAGGAGATCCTCCTTGAACTGTCGCTCGTGTTCAAGATCAAGGAATTCGCACCTGTGTGAAATTGTTATCCGCTCAGA。
partial sequence of Spodoptera frugiperda nucleopolyhedrovirus Hub lef-8 gene:
GGCGGTTCGTACGCAGAATTTTTTCTGTGCCGCGATGTTAAACTGCCCAGTGTCGATTATGAATCTGTAGCTGAAAAGTTTAAATCTTTGTTGGCTGCCAATCTCATCAAAATCGCTTCCTACGACAACGAAGACAACGTTTTAATAGCGTTTAATAATCGTCCTACGATCTTTGAATGCGATAGAGACAAAATGATCTACATCATATATCAGTTGAAACGTAACAAGTTTCCCATTGAAATTAAATTTCACAACAACATTTTGTTTCTGAACCATCACGAAGGTATGATCTGTATAAAGAAAAGATTAAAGATTAACAGCGATGTGGGAATCGTAAAAATAATGACATTGTTGACGCCGTACGAATATCACAATGAATTGTCGTACTTGCGAACCGTGCCCGGCATCTGCGTCGAAGAAGAGAAACATTGTTCGCAATTAATGTCCAAACTGTTACACTACTATTACAAAGATATACTATCCATGTTTGCTACGACGCCGGTGCCCAAGTCTATAGTTTCGTTGACCAATTTGAAAAACGCCATGCCTGTCGTCGCGTACAATAATCTGGGCAACAACGATATATTTTTGGACAATTTACCGGCAGGCAACAGCGTTGTCGTCGCTCCCGAGATTATGCGCAACGACAAAATGTTTCGTTTGTGGACACTGGTGCGCGATCATAAACTGATGACCGCCGAAGACCCCTACATGTGTGAAATTGTTAATCCGCTCAAACTGAAATAACAAA。
partial sequence of Spodoptera frugiperda nucleopolyhedrovirus Hub lef-9 gene:
ATAGGCAACTTTTATACTCTACGTTCTTGACACGGCATCGTTTTACGCAAACGTGCAATGTCTGAACGCGCCCAACGTCGTAGTGCCGCCGAAAAGCAGCATTCAACGATACTACGGCCGCGACGTGGACAACGTTCGCGCTTGGACGACTCGTCATCCCAACATTTCGCAGTTGAGCACCCAAGTTTCCGACGTGCTCCAGCCGAAAAACAACACCGACTGGAGCGTCAAAGTAGGCCTAGGCATTTTCACCGGCGCCAACACCGACTGCGACGGAGACAAGTGTGAAATTGTTATCCGCTCAAA。
the nucleotide sequences of polh, lef-8 and lef-9 genes are found to have more than 99 percent of sequence consistency with the corresponding genes of spodoptera frugiperda nuclear polyhedrosis virus Colombian strain published in GenBank, and the obtained baculovirus is determined to be spodoptera frugiperda nuclear polyhedrosis virus and is named SfMNPV-Hub. The virus strain is preserved in China center for type culture Collection (CCTCC for short, Wuchang Lojia mountain in Wuhan city, Hubei province) in 9 months and 3 days in 2020, and the preservation number is as follows: CCTCC NO: V202054.
Example 2
The method for separating the wild Spodoptera frugiperda nuclear polyhedrosis virus genotype comprises the following specific steps:
1) subjecting Spodoptera frugiperda to nuclear polyhedrosisThe virus SfMNPV-Hub is diluted to 1 × 107,3×106,1×105,1×104And 1X 1035 concentrations of PIB/mL, infecting Spodoptera frugiperda 4-instar by liquid drop method until all the larvae die or pupate, and calculating by probit regression to make the concentration of 5% of the larvae die (LC5) of Spodoptera exigua to be 1.03 × 103PIB/mL, SfMNPV-Hub with the concentration is used for massively infecting 4-year-old beet armyworm larvae, each end of the larvae which are killed by the virus is separately collected, and virus inclusion bodies are purified; infecting 4-instar spodoptera frugiperda larvae with the LC5 concentration of the purified virus inclusion bodies, and collecting the inclusion bodies by single worms; this step was repeated a total of 3 times.
2) The inclusion bodies collected from the single worms were subjected to extraction of viral DNA according to the step (4) of example 1, digestion with PstI, and separation of the digested fragments by agarose gel electrophoresis according to the step (5) of example 1 to give two different genotypes, which were designated SfMNPV-Hub1 and SfMNPV-Hub2, respectively. FIG. 3 shows PstI restriction electrophoretograms of Spodoptera frugiperda nuclear polyhedrosis viruses SfMNPV-Hub, SfMNPV-Hub1 and SfMNPV-Hub2, and lanes 1, 2 and 3 show SfMNPV-Hub, SfMNPV-Hub1 and SfMNPV-Hub2, respectively.
Example 3
The biological activity assay of wild Spodoptera frugiperda nucleopolyhedrovirus and genotypes comprises the following steps:
spodoptera frugiperda nuclear polyhedrosis viruses SfMNPV-Hub, SfMNPV-Hub1 and SfMNPV-Hub2 are all diluted to 1.2X 106,2.4×105,4.8×104,9.6×103And 1.92X 1035 concentrations of PIB/mL, infecting Spodoptera frugiperda 2 at the beginning of its life by the liquid drop method until all the insects die or pupate, and calculating their half-lethal concentration to beet armyworm larvae by using probit regression (LC 50). As a result, it was found that the biological activities of SfMNPV-Hub1 and SfMNPV-Hub were comparable, while the activity of SfMNPV-Hub2 was about 5-fold lower than that of SfMNPV-Hub (Table 1).
TABLE 1 comparison of Semitai lethal concentrations of Spodoptera frugiperda Nuclear polyhedrosis Virus wild type and genotype on beet armyworm larvae
A statistical difference in the median lethal concentration of the two viruses if the 95% confidence limit of the titer does not include the value 1; and vice versa.
Example 4
The method for comparing the yield of wild Spodoptera frugiperda nucleopolyhedrovirus with that of genotype monozoovirus comprises the following specific steps:
by 1X 107SfMNPV-Hub, SfMNPV-Hub1 and SfMNPV-Hub2 of PIBs/mL are respectively infected with 4-year-old beet armyworm, and virus-infected dead larva is collected, diluted to 1mL by distilled water, and counted by a blood counting chamber after being diluted by 1000 times. As shown in FIG. 4, the yield of SfMNPV-Hub1 was 40.8% higher than that of SfMNPV-Hub, while the yield of SfMNPV-Hub2 was 33.7% lower than that of SfMNPV-Hub.
By combining the biological activities and virus yields of wild type and different genotypes, SfMNPV-Hub1 is an ideal production strain for producing spodoptera frugiperda virus insecticides, and can also be widely applied to control spodoptera frugiperda.
The invention is not limited solely to that described in the specification and embodiments, and additional advantages and modifications will readily occur to those skilled in the art, so that the invention is not limited to the specific details, representative embodiments, and illustrative examples shown and described herein, without departing from the spirit and scope of the general concept as defined by the appended claims and their equivalents.
Claims (7)
1. A production strain of a Spodoptera frugiperda virus-resistant insecticide is characterized by being obtained by cloning Spodoptera frugiperda nuclear polyhedrosis virus Hub by polypide, wherein the Spodoptera frugiperda nuclear polyhedrosis virus Hub is preserved in China center for type culture collection (CCTCC NO: V202054) in 9 months and 3 days in 2020.
2. The method for producing a spodoptera frugiperda virus-resistant insecticide producer strain according to claim 1, wherein:
s1, infecting Spodoptera frugiperda with Spodoptera frugiperda nuclear polyhedrosis virus Hub, collecting each virus-lethal larva independently, and purifying virus inclusion bodies;
s2, repeating the step of S1 for two times;
s3, extracting the inclusion body virus DNA, separating and carrying out active screening to obtain a production strain.
3. The method for producing a Spodoptera frugiperda virus-resistant insecticide producer as claimed in claim 2, wherein said infection step is carried out by a liquid-drop method, and the Spodoptera frugiperda nuclear polyhedrosis virus Hub concentration is a concentration that causes 5% mortality.
4. The method for producing a spodoptera frugiperda virus-resistant insecticide-producing strain as claimed in claim 2, wherein said spodoptera frugiperda is spodoptera frugiperda of early 4 years old.
5. The method for producing a Spodoptera frugiperda virus-resistant insecticide producer as claimed in claim 2, wherein said separation step comprises cleavage with PstI, and separation of the cleaved fragments by agarose gel electrophoresis.
6. The method for preparing a spodoptera frugiperda virus-resistant insecticide producer as claimed in claim 2, wherein the activity screening step comprises infecting spodoptera frugiperda 2 at its 2 nd age by a liquid drop method until all the larvae die or pupate, calculating half lethal concentration LC50 on spodoptera exigua larvae by using probit regression, and taking a virus type with low LC50 as the producer.
7. Use of the spodoptera frugiperda virus-resistant insecticide producer strain of claim 1 for controlling spodoptera frugiperda.
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