CN116042539B - Virus for preventing and controlling spodoptera frugiperda, composition and application - Google Patents
Virus for preventing and controlling spodoptera frugiperda, composition and application Download PDFInfo
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- CN116042539B CN116042539B CN202211494482.1A CN202211494482A CN116042539B CN 116042539 B CN116042539 B CN 116042539B CN 202211494482 A CN202211494482 A CN 202211494482A CN 116042539 B CN116042539 B CN 116042539B
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- 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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
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- C12N2710/14011—Baculoviridae
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- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
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Abstract
The invention discloses an insect virus for preventing and controlling spodoptera frugiperda, which is preserved in China Center for Type Culture Collection (CCTCC) at 1 month 18 of 2022, has a preservation number of CCTCC NO: V202202, has a preservation address of China university of Wuhan, is named as nuclear polyhedrosis virus NPV-CS16, can be singly or jointly applied to preventing and controlling spodoptera frugiperda with remarkable effect, and opens up a new way for biological prevention and control of spodoptera frugiperda.
Description
Technical Field
The invention relates to the technical field of spodoptera frugiperda control, in particular to a virus, a composition and application for controlling spodoptera frugiperda.
Background
Insect viruses refer to viruses that host and are pathogenic to insects, including baculoviruses, poxviruses, reoviruses, and the like. Among them, nuclear polyhedrosis virus (nuclear polyhedrosis viruses, NPV) is a virus which was found earlier in insect baculovirus and studied in more detail, and which can proliferate in the nucleus of insect cells, with protein inclusion bodies. Among known insect viruses, the number of species of NPV is the first, and at present, a variety of NPV viral pesticides have been internationally used for controlling agricultural and forestry pests, such as canada, united states and russia for controlling the pine pest gypsy moth with the gypsy moth nuclear polyhedrosis virus; the Brazil is used for preventing and controlling crop pests such as spodoptera frugiperda, spodoptera frugiperda and the like by using a spodoptera frugiperda nuclear polyhedrosis virus (ANTICARSIA GEMMATALIS MNPV, AGMNPV) and a spodoptera frugiperda nuclear polyhedrosis virus (Spodoptera frugiperda MNPV, sfMNPV); NPV such as Medicago sativa nocturnal polyhedrosis virus, helicoverpa Zea MNPV (HzMNPV), helicoverpa virens Nuclear polyhedrosis virus (Heliothis VIRESCENS NPV, HVNPV), aphis citrifolia Nuclear polyhedrosis virus (SYNGRAPHA FALCIFERA MNPV, SFMNPV) and the like are used for preventing and controlling various lepidoptera pests of cotton and vegetables, such as Medicago sativa noctuid, helicoverpa Zea, helicoverpa armigera and the like; cabbage looper nuclear polyhedrosis virus (Mamestra brassicae NPV, mbNPV) and the like are used for controlling looper pests on vegetables.
One of the most successful examples of application of nuclear polyhedrosis virus pesticides is the Brazilian Spodoptera frugiperda nuclear polyhedrosis virus, which is applied to soybeans in approximately 100 tens of thousands of hectares. There are 8 kinds of nuclear polyhedrosis viruses in China which enter field experiments or applications, wherein the application areas are HaNPV, spltNPV, epNPV, bsNPV, grNPV and SeNPV. The virus preparation is produced on a large scale by artificially feeding host insects in China, and SpltNPV, haNPV and the like have been successfully achieved.
Therefore, how to provide an insect virus for preventing and controlling spodoptera frugiperda and to apply the same is a problem to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a virus nuclear polyhedrosis virus NPV-CS16 for preventing spodoptera frugiperda, which can effectively kill spodoptera frugiperda and has good effect on preventing and treating spodoptera frugiperda when being combined with trichogramma.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
A virus for preventing and controlling spodoptera frugiperda is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of V202202 and the preservation address of China university of Wuhan and named as nuclear polyhedrosis virus NPV-CS16 at 1 month 18 of 2022.
As the invention concept same as the technical scheme, the invention also claims the application of the nuclear polyhedrosis virus NPV-CS16 in preparing a preparation for preventing and controlling spodoptera frugiperda.
Preferably, the nuclear polyhedrosis virus NPV-CS16 increases the mortality rate of spodoptera frugiperda and decreases the survival rate and fertility of adults.
As the same inventive concept as the above technical scheme, the present invention also claims a formulation for controlling spodoptera frugiperda, which comprises nuclear polyhedrosis virus NPV-CS16.
Preferably, the formulation further comprises trichogramma.
As the same invention conception as the technical scheme, the invention also claims the application of the combination of the nuclear polyhedrosis virus NPV-CS16 and trichogramma in preventing and controlling spodoptera frugiperda.
As the same inventive concept as the above technical scheme, the present invention also claims a method for controlling spodoptera frugiperda by spraying the virus liquid of the nuclear polyhedrosis virus NPV-CS16 directly onto the surface of crops or directly acting on the surface of the larvae of spodoptera frugiperda, and infecting the larvae of target pests through mouth or wound to cause death thereof.
Preferably, the virus liquid of the nuclear polyhedrosis virus NPV-CS16 is sprayed on tussah eggs or rice moth egg cards which are parasitized by trichogramma, when the trichogramma bites into egg shells to come out after eclosion, the body surface carries the nuclear polyhedrosis virus NPV-CS16, and in the process of searching for eggs to be controlled, viruses are transmitted among target pest populations through probing and ovipositor thorns.
Compared with the prior art, the invention discloses the virus NPV-CS16 for preventing and controlling spodoptera frugiperda, which can effectively kill spodoptera frugiperda and has good effect on preventing and controlling spodoptera frugiperda when being combined with trichogramma.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a view showing an NPV-CS16 microscopic scan provided by the invention;
FIG. 2 is a view showing microscopic scanning of NPV-CS16 virus provided by the invention;
FIG. 3 is a drawing showing an NPV-CS16 transmission electron microscope provided by the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 separation and purification of Spodoptera frugiperda nuclear NPV-CS16
The laboratory screens a nuclear polyhedrosis virus with higher insecticidal activity from a field morbid spodoptera littoralis larva corpse, is named as a spodoptera littoralis nuclear polyhedrosis virus strain NPV-CS16, and observes the ultrastructure of the NPV-CS16 through a scanning electron microscope and a transmission electron microscope
Placing the collected larva corpse into a sterilized mortar, adding a proper amount of sterilized distilled water for grinding, centrifuging at 800r/min by using an ultracentrifuge for 5min, discarding the precipitate, centrifuging the supernatant at 4000r/min by using a centrifuge for 20min, discarding the supernatant, adding a proper amount of sterilized distilled water into the precipitate for suspending, and repeatedly centrifuging for 2-3 times until an off-white precipitate is obtained, and adding a proper amount of sterilized distilled water for suspending the precipitate. For further purification, the suspension of off-white precipitate is added to a prepared sucrose gradient solution with the percentage concentration of 30-60%, centrifuged at 8000r/min for 45min, the white band at 55-60% is recovered, sterilized distilled water is added for dilution, the precipitate is centrifugally recovered, the sterilized distilled water is washed for 3 times, the precipitate is suspended in the sterilized distilled water and is stored at 4 ℃ or minus 20 ℃. The surface of the NPV-CS16 polyhedron is smooth and flat under the scanning electron microscope, the shape is an irregular polyhedron structure, the plane of the NPV-CS16 polyhedron is in various shapes such as quadrangle, pentagon, hexagon and approximate sphere, the size of the polyhedron is different, the diameter of the polyhedron is about 1.0-2.5 mu m, and the average diameter is 1.35 mu m (figures 1 and 2).
The number and arrangement of the virus particles in the NPV-CS16 virus beam can be clearly seen by observing the cross section and the longitudinal section of the virus beam under a transmission electron microscope, the NPV-CS16 virus particles are multi-particle embedded type), a plurality of baculovirus particles are arranged in each inclusion body, the sizes of the baculovirus particles are basically consistent, the virus particles are about 260-400nm long, the diameters of the virus particles are about 45-50nm, and each virus particle capsule contains 1-12 nucleocapsids.
The NPV-CS16 purified strain is obtained by adopting an ultracentrifuge differential centrifugation method in the test. The NPV-CS16 is observed to be in various shapes such as trapezoids, pentagons, hexagons and approximate circles by a scanning electron microscope, the sizes of the trapezoids are different, and the average diameter of the polyhedron is generally about 1.35 mu m. A plurality of virus particles with substantially uniform sizes, about 260nm in length and about 50nm in width, and 1-12 nucleocapsids within each virion envelope were observed under a transmission electron microscope in each inclusion body of NPV-CS16. The virus NPV-CS16 is preserved in China Center for Type Culture Collection (CCTCC) at 1 month and 18 days in 2022, and has a preservation number of CCTCC NO: V202202, a preservation address of university of Chinese, wuhan and is named as nuclear polyhedrosis virus NPV-CS16.
EXAMPLE 2 pathogenicity of NPV-CS16 against 2 noctuid pests
Spodoptera frugiperda: and (3) artificially feeding the insects for more than 3 generations indoors, selecting healthy individuals as healthy insect sources for experiments, and feeding the healthy insect sources with artificial feed to the required age to serve as tested insects.
Cotton bollworm: and (3) artificially breeding and passaging for more than 3 generations indoors, and selecting healthy insect sources as test insects.
The test was carried out in a room-temperature artificial climate incubator (26.+ -. 1 ℃). Healthy larvae of spodoptera frugiperda and cotton bollworms with the same size and 2,3 and 4 ages are selected to be placed in 12 pore plates, 1 head larva is placed in each pore, NPV-CS16 crude extract of 1.0X10 7 PIB/mL is used for respectively infecting the larvae of spodoptera frugiperda and cotton bollworms in each age of 2-4 ages in a room through an artificial feed mixed toxicity method, each treatment is carried out for 3 plates, 4 times of repetition is carried out, meanwhile, fresh and nontoxic artificial feed is used as blank control, the death number is investigated every day, and the death rate is calculated.
The results were as follows:
(1) Toxicity determination of NPV-CS16 on two kinds of noctuid larvae in different ages
After the spodoptera frugiperda larvae of different ages are fed with the virulent NPV-CS1624h, the death number of the larvae of different ages is tracked and observed every day until the 10d, and the research result shows that the early feeding of the spodoptera frugiperda larvae of 2 and 3 ages reaches the death peak period at the 5 d; the early feeding of spodoptera littoralis larvae at 4 years followed by NPV-CS16 reached the peak of death on day 6. The average death number of larvae of spodoptera frugiperda larvae at different ages after NPV-CS16 feeding is respectively 32, 30 and 23, which are respectively increased by 30, 27 and 22 compared with the control group.
After feeding the cotton bollworm larvae of different ages for NPV-CS1624 hours, tracking and observing the death numbers of the larvae of different ages every day until 10d, and finding that the death peak period can be reached at 5d after the initial feeding of the cotton bollworm larvae of 2 and 3 ages for NPV-CS 16; the average larval death number of the 4-year-old cotton bollworms after the 4-year-old cotton bollworms ingest NPV-CS16 reaches the death peak period in the 7d, and the average larval death numbers of the cotton bollworms at different ages after the 4-year-old cotton bollworms ingest NPV-CS16 are respectively 30, 28 and 20, and 27, 25 and 18 of the cotton bollworms are respectively increased compared with the control group.
(2) Influence of NPV-CS16 on mortality of two noctuid larvae of different ages
2. Spodoptera frugiperda and cotton bollworm larvae of ages 3 and 4 die substantially after ingestion of NPV-CS 16. The average mortality of the larvae of spodoptera frugiperda and cotton bollworms at different ages after 10 days of NPV-CS16 virus intake is significantly higher than that of the control group (P < 0.05); the research result shows that the pathogenicity of NPV-CS16 to spodoptera frugiperda and cotton bollworm 2, 3-year-old larvae is obviously higher than that of 4-year-old larvae; the average mortality rate between spodoptera frugiperda and 2, 3-instar larvae of cotton bollworms is not obviously different (P is more than 0.05), but is obviously different from that of 4-instar larvae (P is less than 0.05), so that the pathogenicity of NPV-CS16 virus gradually decreases along with the growth of 2 kinds of spodoptera insect ages; corrected mortality of spodoptera frugiperda 2,3, 4 instar larvae was 87%, 81% and 63%, respectively; the corrected mortality rates for the larvae of cotton bollworms 2,3, and 4 were 81%, 75%, and 53%, respectively.
Mechanism of action: the virus that the NPV enters the insect body orally is digested by gastric juice, and baculovirus particles are released, enter the body cavity through the midgut epithelial cells, invade the cells, proliferate in the nucleus, and then invade the healthy cells until the insect is fatal. The faeces and dead insects are used for infecting other insects, so that virus diseases are prevalent in pest populations, and the pest harm is controlled.
(3) Influence of NPV-CS16 on longevity and fertility of two noctuid adults
After NPV-CS16 is fed to spodoptera litura and cotton bollworm adults on day 1, the lives of male and female adults of the two spodoptera litura pests are not obviously influenced (P is more than 0.05). The lives of male and female adults after the spodoptera frugiperda initially eclosion of male and female adults and the feed of NPV-CS16 are respectively 9.4d and 11.4d, and compared with a control group, the lives of the male and female adults are respectively shortened by 0.36 d and 0.42d; the lives of male and female adults after the initial emergence of the cotton bollworms and the inoculation of the toxin NPV-CS16 are respectively 10.5 days and 8.54 days, and compared with a control group, the lives of the male and female adults are respectively shortened by 0.30 days and 0.18 days.
After the first eclosion spodoptera frugiperda and adult cotton bollworms are fed with 10% honey water containing NPV-CS16 on day 1, NPV-CS16 is found to have a significant effect on the egg laying amount of the adult spodoptera frugiperda (P < 0.05), but no significant effect on the egg laying amount of the adult cotton bollworms (P > 0.05). After the female adult spodoptera frugiperda is eclosion and is fed with the virulent NPV-CS16 on the 1 st day, the average spawning amount of female moths is 543 grains/female, and is reduced by 108 grains/female compared with the average spawning amount of female moths in a control group; after the adult cotton bollworms are eclosion and fed with the virulent NPV-CS16 on day 1, the average spawning quantity of female moths is 589 grains/female, and the average spawning quantity of female moths is reduced by 51 grains/female compared with that of female moths in a control group.
After the first eclosion spodoptera frugiperda and the adult cotton bollworms are fed with the virulent NPV-CS16 on day 1, the hatching rate of the female moths of the two kinds of spodoptera frugiperda insects is not obviously affected (P is more than 0.05). The hatching rate of female moths after the adult spodoptera litura of the initial eclosion meets the poison NPV-CS16 is 83%, and the hatching rate of eggs is reduced by 2.0% compared with a control group; the hatching rate of female moths spawning after the adult cotton bollworms of the initial eclosion meet the toxin NPV-CS16 is 82 percent, and is reduced by 5 percent compared with a control group.
Example 3 indoor control Effect of NPV-CS16 on Spodoptera frugiperda by combination of trichogramma
Test insects
The spodoptera littoralis to be tested is provided by a comprehensive prevention and control laboratory of economic crop diseases and insect pests of the Hunan agricultural university plant protection institute, and is tested after the indoor artificial feed is continuously fed for a plurality of generations. Fresh full single-layer egg blocks initially produced within 8 hours are selected for testing, and for interference elimination, the tested eggs are uniformly treated with 5% formaldehyde solution for 10 seconds for standby before testing. Trichogramma and trichogramma borer are put into an insect-raising box with air permeability for raising, after eclosion, a proper amount of full inactivated tussah eggs are added into the insect-raising box for parasitic trichogramma and are continuously raised for multiple generations in an artificial climate incubator. The feeding conditions are as follows: 26±1 ℃, relative Humidity (RH): 40% -50%, photoperiod: l// D = 14h//10h.
Test virus NPV-CS16
Indoor control test of spodoptera frugiperda by combination of NPV-CS16 and two trichogramma
Picking fresh full single-layer egg mass without coating which is initially produced in 8h of spodoptera frugiperda, placing the egg mass into a same vessel, soaking the egg mass in 5% formaldehyde solution for 10s for egg surface sterilization, then washing the egg mass with sterile water for several times, then soaking the egg mass in 2.0X10 7 PIB/mL NPV-CS16 virus liquid for 10s, and sucking redundant virus liquid on the surface of the egg mass by using filter paper. After the egg blocks are naturally dried at room temperature, split charging the egg blocks into transparent insect-raising boxes according to the bee egg ratio of 1:5, inoculating trichogramma and trichogramma borer, additionally arranging ovum blocks with 5% formaldehyde solution for ovum surface disinfection as control,
Influence of NPV-CS16 on hatching rate of spodoptera frugiperda eggs by combination of two trichogramma species
After the disinfection treatment by using 5% formaldehyde solution, the parasitic egg masses of trichogramma and trichogramma borer are removed after 24 hours, and the egg hatching rate of spodoptera frugiperda is only 32% and 36%. The hatching rates of spodoptera frugiperda eggs in the NPV-CS16+ pine moth trichogramma combined group and the NPV-CS16+ stem borer trichogramma combined group are respectively 34% and 39%, and the results show that the inhibition effects of the independent parasitic treatment of the pine moth trichogramma and the stem borer trichogramma or the combination of the NPV-CS16 on hatching of spodoptera frugiperda are not obviously different (P is more than 0.05). Indoor experiments of the virus prove that the virus is harmless to trichogramma; egg hatching is not prominent, indicating that NPV-CS16 is not primarily directed to egg stage, it is primarily directed to larvae, and the combined group is slightly more lethal to larvae than the individual group.
NPV-CS16 and two trichogramma combined use have lethal effect on spodoptera frugiperda larvae
After follow-up observation of hatched larvae, the larvae of the combined group of NPV-CS16 and trichogramma were found to die before 3 years. The mortality rate of larvae of the trichogramma of NPV-CS16+ pine caterpillar, the trichogramma of NPV-CS16+ borer and the spodoptera frugiperda of the NPV-CS16 treated group is obviously higher than that of the control group in 5% formaldehyde solution, and the larvae correction mortality rates of the trichogramma of NPV-CS16+ pine caterpillar and the trichogramma of NPV-CS16 treated group are respectively 80%, 77% and 66% without obvious difference. Studies have shown that the above treatments all have a significant poisoning effect on spodoptera frugiperda larvae (P < 0.05).
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (8)
1. A virus for preventing and controlling spodoptera frugiperda is characterized in that the virus is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of V202202 and the preservation address of China university of Wuhan and named as nuclear polyhedrosis virus NPV-CS16 in 1 month 18 of 2022.
2. Use of the nuclear polyhedrosis virus NPV-CS16 of claim 1 for the preparation of a formulation for controlling spodoptera frugiperda.
3. The use according to claim 2, wherein the nuclear polyhedrosis virus NPV-CS16 increases mortality of spodoptera frugiperda and decreases survival and fertility of adults.
4. A formulation for controlling spodoptera frugiperda, said formulation comprising the nuclear polyhedrosis virus NPV-CS16 of claim 1.
5. A formulation for controlling spodoptera frugiperda according to claim 4 and further comprising trichogramma.
6. Use of the nuclear polyhedrosis virus NPV-CS16 according to claim 1 in combination with trichogramma for controlling spodoptera frugiperda.
7. A method for controlling spodoptera frugiperda, characterized in that the virus liquid of the nuclear polyhedrosis virus NPV-CS16 of claim 1 is sprayed directly onto the surface of crops or directly acts on the surface of the larvae of spodoptera frugiperda to infect target pests through mouth or wound, thereby causing death thereof.
8. A method for controlling spodoptera frugiperda according to claim 7 wherein the virus liquid of the nuclear polyhedrosis virus NPV-CS16 of claim 1 is sprayed on tussah eggs or rice moth egg cards which have been parasitized by trichogramma, and when trichogramma bites into eggs and comes out, the body surface carries the nuclear polyhedrosis virus NPV-CS16 of claim 1, and in the process of searching eggs to be controlled, viruses are spread among target pest populations by probing and ovipositor thorns.
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