CN111117971A - Domestication method and application of nuclear polyhedrosis virus suitable for yellow poplar silk wild borer - Google Patents
Domestication method and application of nuclear polyhedrosis virus suitable for yellow poplar silk wild borer Download PDFInfo
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- CN111117971A CN111117971A CN202010057058.5A CN202010057058A CN111117971A CN 111117971 A CN111117971 A CN 111117971A CN 202010057058 A CN202010057058 A CN 202010057058A CN 111117971 A CN111117971 A CN 111117971A
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- 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
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Abstract
The invention discloses a domestication method and application of nuclear polyhedrosis virus suitable for yellow poplar silk wild borer. The domestication method comprises the following steps: diluting the nuclear polyhedrosis virus by 10-20 times with water, and applying the diluted nuclear polyhedrosis virus to branches and leaves of Buxus microphylla to obtain a virus-containing feed; treating yellow poplar wild borer larva with matrine, feeding hungry yellow poplar yellow mulberry wild borer larva with virus containing feed, collecting virus-infected or dead larva, crushing, filtering, centrifuging the filtrate, collecting and sterilizing to obtain domesticated free nuclear polyhedrosis virus; taking the protein as a poison source, repeating the steps, and increasing domestication times; and culturing and amplifying the domesticated free nuclear polyhedrosis virus to obtain the nuclear polyhedrosis virus. The nuclear polyhedrosis virus has strong killing performance on the yellow poplar wild borer, has the characteristics of strong pertinence, long control time and difficulty in generating drug resistance of pests, and has high domestication success rate.
Description
Technical Field
The invention relates to a nuclear polyhedrosis virus domestication and preparation method, in particular to a nuclear polyhedrosis virus domestication and industrial production method and application thereof suitable for yellow poplar, silk moth, and belongs to the technical field of biological pesticides.
Background
The yellow poplar silk borer, Diaphania perspectalis (Walker), an insect belonging to the genus Diaphania of the family Bombycidae of Lepidoptera, is widely distributed. It mainly harms plants of Buxus, such as Buxus sempervirens, Buxus tremuloides, Buxus megalophylla, Buxus microphylla, Buxus koreana, and Chinese ilex, Celastrus angulatus, etc., wherein the most serious damage is caused by Buxus sempervirens and Buxus tremuloides. The young plants and leaves are eaten by larvae, the young plants and leaves are usually spinned and conjugated, the larvae eat the larvae and the leaves, the damaged leaves are scorched, the rate of the serious street damaged plants is over 50 percent and even can reach 90 percent, the leaves can be eaten by light during outbreak, the adult plants of the Chinese littleleaf poplar die, the appearance of the market is influenced, and the environment is polluted.
Chemical drug control is still an important emergency measure for controlling the insects, the control drugs are mainly organophosphorus pesticides, and are also applied to abamectin and BT emulsions, but the problems of increased drug resistance, serious damage of natural enemies, environmental pollution and the like are generally encountered, and particularly, the thorough control is difficult to achieve due to generation overlapping.
In 1973, the united nations food and agriculture organization and the world health organization recommended insect baculovirus for biological control of crop pests. Insect virus is one of the important means for biological control, and has the advantages of strong specificity, high toxicity, high stability, safety, no harm, capacity of causing epidemic spread of pest group virus diseases after use, and capacity of naturally controlling pest growth and pest elimination in a long time, resulting in continuous toxic infection and death of successive generations. Compared with other chemical pesticides and biological pesticides, the insect virus pesticide has no resistance problem to be found so far, which brings good opportunity for the development of virus pesticides. The major components currently used in biological control are baculoviruses, and particularly nuclear polyhedrosis viruses.
The existing insect virus sources are mainly found and collected in the field, but the method is expensive, high in cost, time-consuming and low in efficiency, and cannot meet the requirements of outbreak, prevention and control of insect conditions in modern environments.
Disclosure of Invention
The invention mainly aims to provide a domestication method and application of nuclear polyhedrosis virus suitable for yellow poplar silk wild borer, so as to overcome the defects of the prior art.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
the embodiment of the invention provides a method for domesticating nuclear polyhedrosis virus suitable for yellow poplar silk wild borer, which comprises the following steps:
1) providing nuclear polyhedrosis virus, adding water to dilute by 10-20 times, and applying to branches and leaves of Buxus microphylla to obtain a virus-containing feed;
2) treating the yellow poplar silk moth larvae with matrine, feeding the starved yellow poplar silk moth larvae with the virus-containing feed, collecting the virus-infected insects or dead insects, crushing, filtering, centrifuging the filtrate, collecting, and sterilizing to obtain domesticated free nuclear polyhedrosis virus;
3) taking the domesticated free nuclear polyhedrosis virus as a virus source, repeating the steps 1) to 2), and increasing domestication times;
4) and (3) carrying out insect body culture and amplification on the domesticated free nuclear polyhedrosis virus obtained in the step 3), and then purifying to finally obtain the nuclear polyhedrosis virus suitable for the yellow poplar wild borer.
Further, the nuclear polyhedrosis virus includes, but is not limited to, Autographa californica nuclear polyhedrosis virus, Ectropis obliqua nuclear polyhedrosis virus, Spodoptera litura nuclear polyhedrosis virus, or Spodoptera exigua nuclear polyhedrosis virus, etc.
Further, the domestication generation in the step 3) is 20-30 generations.
The embodiment of the invention also provides the nuclear polyhedrosis virus which is prepared by the domestication method and is suitable for the yellow poplar wild borer.
The embodiment of the invention also provides a biological pesticide, and the effective component of the biological pesticide comprises the nuclear polyhedrosis virus suitable for the yellow poplar silk wild borer.
Further, the biological pesticide also comprises an emulsifier, a dispersant, a thickening agent, an antifreezing agent and an antifoaming agent, and the balance of the biological pesticide comprises water.
Compared with the prior art, the invention has the advantages that:
1) the domesticated nuclear polyhedrosis virus has stronger killing performance on the yellow poplar wild rice borer, the insecticidal rate is more than 90 percent, and the domesticated nuclear polyhedrosis virus has the characteristics of strong pertinence, good dispersibility, uniform dissolution, quick absorption, high drug effect and difficulty in generating drug resistance of pests and has good use effect;
2) before feeding, the larva is treated by a chemical agent (in the case, matrine) with sub-lethal dose in advance, so that the immune system of the larva is destroyed, and viruses are easier to infect;
3) the invention directionally cultures the needed virus by an indoor domestication method, has strong practicability and high domestication efficiency compared with the prior field virus collection preparation and living body culture domestication, and can greatly save time, cost, manpower and material resources.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a photograph showing the harmful state of pests in example 2 of the present invention.
FIG. 2 is a photograph showing death of pests 5 days after application of the drug in effect test example 2 of the present invention.
FIG. 3 is a photograph showing the recovery growth of Buxus microphylla 30 days after administration of the drug in the effect verification example 2 of the present invention.
FIG. 4 is a photograph showing healthy growth of Buxus microphylla 140 days after administration of the drug in the efficacy test example 2 of the present invention.
Detailed Description
In view of the technical problems in the prior art, the inventor provides a technical scheme of the invention through long-term research and massive practice, and the technical scheme is mainly an indoor domestication and preparation method of the nuclear polyhedrosis virus suitable for the yellow poplar wild borer. The method specifically comprises the steps of virus domestication, nuclear polyhedrosis virus collection and amplification, reagent type preparation and the like.
The technical solution, its implementation and principles, etc. will be further explained as follows.
One aspect of the embodiments of the present invention provides a method for acclimatizing nuclear polyhedrosis virus suitable for yellow poplar, which is a method for directionally acclimatizing and cultivating required nuclear polyhedrosis virus by indoor insect culture, and mainly comprises:
1) providing nuclear polyhedrosis virus, adding water to dilute the nuclear polyhedrosis virus by 5-20 times, and applying the nuclear polyhedrosis virus to branches and leaves of Buxus microphylla to obtain a virus-containing feed;
2) firstly, treating the larvae of the yellow-poplar yellow-silk borers by 3000-4000 times of the low dose of the matrine biological pesticide to destroy the immunity of the yellow-poplar yellow-silk borers and improve the infectivity of viruses;
3) feeding hungry euonymus sempervirens larvae with the virus-containing feed, collecting toxigenic or dead insects, crushing, filtering, centrifuging filtrate, collecting, and sterilizing to obtain domesticated free nuclear polyhedrosis virus;
4) taking the domesticated free nuclear polyhedrosis virus as a virus source, repeating the steps 1) to 3), and increasing domestication times;
5) and (3) carrying out insect body culture and amplification on the domesticated free nuclear polyhedrosis virus obtained in the step 3) according to the steps 1) and 3), and then purifying to finally obtain the nuclear polyhedrosis virus suitable for the yellow poplar wild borer.
In some embodiments, step 1) comprises: diluting the nuclear polyhedrosis virus pesticide with sterile water, and soaking branches and leaves of Buxus microphylla.
Further, the volume ratio of the nuclear polyhedrosis virus pesticide to the sterile water is 1: 10 to 20.
Further, the nuclear polyhedrosis virus includes, but is not limited to, Autographa californica nuclear polyhedrosis virus, Ectropis obliqua nuclear polyhedrosis virus, Spodoptera litura nuclear polyhedrosis virus, or Spodoptera exigua nuclear polyhedrosis virus, etc.
In some embodiments, step 2) comprises: placing eggs of the yellow poplar silk borers in a climatic incubator for incubation and colony breeding, and selecting 3-4-year-old yellow poplar silk borer larvae, wherein the culture conditions of the climatic incubator comprise: the temperature is 25.5-26.5 ℃, the humidity is 55-65%, and the ratio of the illumination time to the dark time is 14-12:10 to 12.
Further, step 3) comprises: treating 3-4-year-old yellow poplar wild borer larvae by diluting 3000-4000 times with matrine biological pesticide at low dose, starving for 12-24 hours, feeding the virus-containing feed, feeding normal branches and leaves of the small poplar, observing, and collecting virus-infected insects or dead insects.
In some embodiments, the acclimation generation in step 4) is 20-30 generations.
In some more specific embodiments, the biopesticide virus applied to the control of the yellow poplar, namely the yellow poplar, wild borer is named nuclear polyhedrosis virus, and the domestication method of the virus comprises the following steps:
step one, primary infection of viruses: breeding great amount of yellow poplar stem borer larva indoors, diluting nuclear polyhedrosis virus pesticide purchased from market with sterile water at a low ratio (1: 10-20, equivalent to 50-200 times of normal use concentration), soaking fresh small leaf yellow poplar branch and leaf, and drying in the shade. Selecting 3-4-year-old yellow poplar silk moth larvae, treating the yellow poplar silk moth larvae with matrine, starving the yellow poplar silk moth larvae for a period of time, generally 12-24 hours, feeding the treated branches and leaves of the small leaf yellow poplar, feeding the normal branches and leaves after receiving the toxic feed, observing and collecting poisoned dead or dying larvae, grinding the larvae with a mortar, filtering by 4 layers of gauze, centrifuging, collecting supernatant, and sterilizing to obtain free viruses, namely the free viruses domesticated with the nuclear polyhedrosis viruses of the generation 1, which are used as a virus source for next culture. Wherein the culture conditions of the climatic incubator comprise: the temperature is 26 +/-0.5 ℃, the humidity is 60 +/-5%, and the ratio of the illumination time to the dark time is 14-12:10 to 12.
And step two, treating branches and leaves with the virus obtained in the step one, feeding larvae of the yellow poplar, namely the Phlebopus medinalis, collecting dead or frequently dead larvae, and collecting the virus, namely the free virus for domesticating the 2-generation nuclear polyhedrosis virus.
And step three, repeating the steps, increasing domestication generation times, improving the toxicity of the virus to the yellow poplar, namely the yellow poplar, and accumulating for 20-30 generations.
And step four, culturing and amplifying the domesticated virus after the virus virulence meets the set requirement.
Furthermore, the domestication method provided by the invention is applied to directional domestication of viruses for preventing and treating yellow poplar, silk moth and the like in a laboratory, and is not used for field collection.
Further, the virus indoor directional domestication and cultivation of the invention is applicable but not limited to the application on the yellow poplar, sericin indicathea.
Multiple times of pharmacodynamic tests prove that the domesticated nuclear polyhedrosis virus has the characteristics of strong pertinence, good dispersibility, uniform dissolution, quick absorption, high efficacy and difficulty in generating drug resistance of pests on the yellow poplar wild rice borer. Compared with the existing field virus collection and preparation, the method has strong practicability, can greatly save time, cost and labor force, has no toxic or side effect on a human ecological circulation system and crops, has a good promotion effect on the development of organic agriculture, and has important economic and social meanings.
In another aspect of the embodiments of the present invention, there is provided a nuclear polyhedrosis virus adapted to yellow poplar, silk moth, prepared by the acclimatization method.
In another aspect of the embodiments of the present invention, there is provided a biopesticide for yellow poplar wild borer, wherein the active ingredient of the biopesticide comprises the above nuclear polyhedrosis virus for yellow poplar wild borer.
In some embodiments, the biopesticide further comprises an emulsifier, a dispersant, a thickener, an antifreeze, an antifoaming agent, and the balance comprises water.
The inventors also verified the control effect of the nuclear polyhedrosis virus prepared by the above method:
1) the control object is: experiments prove that the domesticated nuclear polyhedrosis virus has a remarkable control effect on the yellow poplar wild rice borer.
2) The nuclear polyhedrosis virus domesticated by the invention can not generate drug resistance after being used, does not hurt natural enemies, is safe to people, livestock and other organisms, and has long prevention and treatment time.
In conclusion, the domesticated nuclear polyhedrosis virus has a strong control effect on the yellow poplar yellow silk moth, the killing rate is more than 90%, and the nuclear polyhedrosis virus has the effects of long control time, high control efficiency and no damage to natural enemies; the invention directionally cultures the needed virus by an indoor domestication method, has strong practicability and high domestication efficiency compared with the prior field virus collection preparation and living body culture domestication, and can greatly save time, cost, manpower and material resources.
The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and several embodiments. However, the examples are chosen only for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
Comparative example 1
The infection promotion treatment of the matrine to the nuclear polyhedrosis virus:
the biopesticide virus applied to the infection treatment of the yellow poplar silk moth is named as autographa californica nuclear polyhedrosis virus, and the treatment comprises the following steps:
(1) cleaning fresh leaves of Buxus microphylla, drying in the shade, spraying 1000, 2000, 3000, 4000 times of 1% matrine wettable powder or clear water (used as control), and drying in the shade; in addition, diluting the autographa californica nuclear polyhedrosis virus pesticide purchased from the market with sterile water at a low ratio (1: 20), soaking fresh populus microphylla branches and leaves, and drying in the shade;
(2) preparing yellow poplar silk wild borer larvae: artificially breeding a large number of yellow poplar, namely, the yellow poplar, the wild borers, and arranging a climatic incubator: temperature 26 + -0.5 deg.C, humidity 60 + -5%, illumination: selecting 3-4-instar chrysosporium chrysosplenium medinalis larvae to starve for 12-24 hours, wherein the darkness is 14: 10;
(3) treating branches and leaves with the matrine obtained in the step (1), feeding normal branches and leaves after feeding larvae of the yellow-poplar, silk moth for 2 hours, treating the branches and leaves with the matrine obtained in the step (1), feeding the branches and leaves with the autographa californica nuclear polyhedrosis virus pesticide for 2 hours, feeding the branches and leaves after one day, and observing the death condition of the larvae in contrast; the relationship between matrine treatment and larva lethality (%) of yellow poplar silk borer is shown in the following table:
| treatment of | Matrine 0 | Matrine 4000 | Matrine 3000 | Matrine 2000 | Matrine 1000 |
| Matrine | 0.00 | 0.00 | 0.01 | 0.17 | 73.25 |
| Matrine + polyhedrin | 0.00 | 0.02 | 1.10 | 8.90 | 78.92 |
Note: matrine 0 is treated with clear water, and matrine 4000 is 4000 times of 1% matrine wettable powder
The comparison example shows that about 2000 times of matrine treatment can not directly cause the death of the diaphania chrysosporium medinalis walker, and simultaneously has a remarkable effect of promoting the infection of the diaphania chrysosporium nuclear polyhedrosis virus, through logistic regression analysis, the immunity of the diaphania chrysosporium medinalis walker can be effectively destroyed, the infection power of the diaphania chrysosporium nuclear polyhedrosis virus is improved, and the matrine use concentration of the matrine for directly killing the diaphania chrysosporium medinalis walker can be effectively avoided to be 1800 and 2400 times, preferably 2000 times.
Comparative example 2
Domestication treatment of cotton bollworm nuclear polyhedrosis virus:
the name of the biopesticide virus applied to the prevention and the treatment of the yellow poplar, namely the cotton bollworm nuclear polyhedrosis virus, and the domestication of the virus and the preparation method of the biopesticide comprise the following steps:
(1) primary infection with virus: feeding a large amount of wild chrysoidea larva, diluting a cotton bollworm nuclear polyhedrosis virus pesticide purchased from the market with sterile water at a low ratio (1: 20), soaking fresh branches and leaves of Buxus microphylla, drying in the shade, selecting 3-4-year-old wild chrysoidea larva, starving for a period of time, generally 12-24 hours, feeding the treated branches and leaves of Buxus microphylla, feeding normal branches and leaves after inoculation feed is consumed, observing and collecting poisoned dead or dying larva, grinding by using a mortar, filtering by using 4 layers of gauze, centrifuging, collecting supernatant, sterilizing to obtain free virus, namely the free virus domesticated with 1 generation nuclear polyhedrosis virus, and using the free virus as a virus source for next culture. Setting a climate incubator: temperature 26 + -0.5 deg.C, humidity 60 + -5%, illumination: 14-12:10-12 in darkness.
(2) Treating branches and leaves with the virus obtained in the step (1), feeding larvae of the yellow poplar, wild borers, collecting dead or frequently dead larvae, and collecting the virus, namely the free virus for domesticating the nuclear polyhedrosis virus of the generation 2.
(3) Repeating the steps, increasing domestication generation times, and improving the toxicity of the virus to the yellow poplar silk wild borer. The relationship between acclimation generation and pathogenic rate is shown in the following table:
this comparative example shows that the Heliothis armigera nuclear polyhedrosis virus has no significant infection effect on Photinia chrysosporium.
Example 1
Acclimatization treatment of prodenia litura nuclear polyhedrosis virus:
the name of the biopesticide virus applied to the prevention and control of the yellow poplar, namely the spodoptera litura nuclear polyhedrosis virus, and the domestication of the virus and the preparation method of the biopesticide comprise the following steps:
(1) primary infection with virus: breeding a large number of chrysoidea silk wild borer larvae, diluting a prodenia litura nuclear polyhedrosis virus pesticide on the market with sterile water at a low ratio (1: 20), soaking fresh branches and leaves of chrysoidea silk borer, drying in the shade, selecting 3-4-year-old chrysoidea silk wild borer larvae, treating the chrysoidea silk borer larvae with matrine, starving for a period of time, generally 12-24 hours, feeding the treated branches and leaves of the chrysoidea silk borer, feeding normal branches and leaves after the inoculation of a virus feed, observing, collecting poisoned or dying larvae, grinding the larvae with a mortar, filtering by 4 layers of gauze, centrifuging, collecting supernatant, and performing acclimation to obtain free viruses, namely the free viruses of the nuclear polyhedrosis viruses of the generation 1, and using the free viruses as a poison source for next culture. Setting a climate incubator: temperature 26 + -0.5 deg.C, humidity 60 + -5%, illumination: 14-12:10-12 in darkness.
(2) Treating branches and leaves with the virus obtained in the step (1), feeding larvae of the yellow poplar, wild borers, collecting dead or frequently dead larvae, and collecting the virus, namely the free virus for domesticating the nuclear polyhedrosis virus of the generation 2.
(3) Repeating the steps, increasing domestication generation times, and improving the toxicity of the virus to the yellow poplar silk wild borer. The relationship between acclimation generation and pathogenic rate is shown in the following table:
example 2
Domestication treatment of beet armyworm nuclear polyhedrosis virus:
the biopesticide virus applied to the prevention and control of the yellow poplar, namely spodoptera exigua nuclear polyhedrosis virus, and the domestication of the virus and the preparation method of the biopesticide comprise the following steps:
(1) primary infection with virus: a large number of chrysosporium larvae are bred, spodoptera exigua nuclear polyhedrosis virus pesticides purchased from the market are diluted by sterile water at a low ratio (1: 10) and then soaked in fresh chrysosporium branches and leaves, the moisture is dried in the shade, 3-4-year-old chrysosporium larvae are selected, the chrysosporium larvae are treated by matrine, after hunger for a period of time, generally 12-24 hours, the treated chrysosporium branches and leaves are fed, normal branches and leaves are fed after virus inoculation is finished, the poisoned and dead or dying larvae are observed and collected, after mortar grinding, 4 layers of gauze are filtered, centrifugation is carried out, supernatant is collected, and bacteria are removed, so that free state viruses, namely the free state viruses domesticated for the nuclear polyhedrosis viruses of 1 generation are obtained and serve as a poison source for next culture. Setting a climate incubator: temperature 26 + -0.5 deg.C, humidity 60 + -5%, illumination: 14-12:10-12 in darkness.
(2) Treating branches and leaves with the virus obtained in the step (1), feeding larvae of the yellow poplar, wild borers, collecting dead or frequently dead larvae, and collecting the virus, namely the free virus for domesticating the nuclear polyhedrosis virus of the generation 2.
(3) Repeating the steps, increasing domestication generation times, and improving the toxicity of the virus to the yellow poplar silk wild borer. The relationship between acclimation generation and pathogenic rate is shown in the following table:
example 3
Acclimatization treatment of autographa californica nuclear polyhedrosis virus:
the name of the biopesticide virus applied to the prevention and the treatment of the yellow poplar, namely the autographa californica nuclear polyhedrosis virus, the domestication of the virus and the preparation method of the biopesticide comprise the following steps:
the name of the biopesticide virus applied to the prevention and the treatment of the yellow poplar, namely the autographa californica nuclear polyhedrosis virus, the domestication of the virus and the preparation method of the biopesticide comprise the following steps:
(1) primary infection with virus: breeding a large number of chrysosporium lucida larvae, diluting nuclear polyhedrosis virus pesticide purchased from the market with sterile water at a low ratio (1: 20), soaking fresh chrysosporium litura branches and leaves, drying in the shade, selecting 3-4-year-old chrysosporium lucida larvae, treating the chrysosporium lucida larvae with matrine, starving for a period of time, generally 12-24 hours, feeding the treated branches and leaves, feeding normal branches and leaves after the inoculation feed is consumed, observing and collecting poisoned and dead or dying larvae, grinding the larvae with a mortar, filtering with 4 layers of gauze, centrifuging, collecting supernatant, sterilizing to obtain free viruses, namely the free viruses domesticated for the nuclear polyhedrosis virus generation 1, and using the free viruses as a poison source for next culture. Setting a climate incubator: temperature 26 + -0.5 deg.C, humidity 60 + -5%, illumination: 14-12:10-12 in darkness.
(2) Treating branches and leaves with the virus obtained in the step (1), feeding larvae of the yellow poplar, wild borers, collecting dead or frequently dead larvae, and collecting the virus, namely the free virus for domesticating the nuclear polyhedrosis virus of the generation 2.
(3) Repeating the steps, increasing domestication generation times, and improving the toxicity of the virus to the yellow poplar silk wild borer. The relationship between acclimation generation and pathogenic rate is shown in the following table:
effect test example 1
(1) And continuing to amplify the virus domesticated for 30 generations by culturing the yellow poplar silk wild borer. Dead or nearly dead insects are collected after general culture for 5-7 days, and the nuclear polyhedrosis virus can be released by ultrasonic pulverization.
(2) The nuclear polyhedrosis virus is purified, differential centrifugation is performed to remove worm fragments and purify the nuclear polyhedrosis virus.
(3) In a quantitative count (1000-12000 billion) of nuclear polyhedrosis viruses: 0.95-1.05 g of emulsifier, 3.95-4.05 g of dispersant, 0.24-0.26 g of defoamer and deionized water to make up to 100 ml, and shaking up to prepare the trial preparation.
(5) Diluting the prepared trial preparation by using deionized water for 1000 times and 1500 times, soaking fresh branches and leaves of buxus microphylla, drying in the shade, selecting 3-4-year-old larvae of the yellow poplar, starving for a period of time, generally 12-24 hours, feeding the treated branches and leaves of the buxus microphylla, feeding normal branches and leaves after the inoculated feed is eaten, and observing and collecting poisoned dead or dying larvae after 10 days. The fatality (%) of the tested preparations of different viruses after domestication and the larvae of the yellow poplar silk moth is shown in the following table:
| treatment of | Spodoptera litura nuclear polyhedrosis virus | Spodoptera exigua nuclear polyhedrosis virus | Autographa californica nuclear polyhedrosis virus |
| Mortality (%) | 80.01 | 84.95 | 91.37 |
The effect verification example shows that the nuclear polyhedrosis virus suitable for biological control of the yellow poplar, namely the agriopogaea virens can be cultured in an artificial domestication mode, and the preferred alfalfa silverweed moth nuclear polyhedrosis virus has a more prominent domestication effect.
Effect test example 2
(1) And continuing to culture the southern yellow poplar wild borer to amplify the 30-generation domesticated Autographa californica nuclear polyhedrosis virus. Dead or nearly dead insects are collected after general culture for 5-7 days, and the nuclear polyhedrosis virus can be released by ultrasonic pulverization.
(2) Purifying the autographa californica nuclear polyhedrosis virus, carrying out differential centrifugation to remove worm fragments and purifying the nuclear polyhedrosis virus.
(3) The addition of the following components in proportion to the alfalfa silver leaf moth nuclear polyhedrosis virus with quantitative counting (1000-12000 hundred million): 0.95-1.05 g of emulsifier, 3.95-4.05 g of dispersant, 0.24-0.26 g of defoamer and deionized water to make up to 100 ml, and shaking up to prepare the trial preparation.
(5) Diluting the prepared trial preparation with water 1000 times, spraying the diluted preparation on Populus tremula (shown in figure 1) with a sprayer 6 month and 9 days 2019, and regularly observing the death of the larvae of the Populus tremula and the growth and recovery conditions of the Populus tremula.
(6) In 6 months and 14 days, the yellow poplar, namely the yellow poplar, is found dead by the wild borers after 5 days of medication (as shown in figure 2), and the newly born branches and leaves of the small poplar are not eaten by pests any more in 7 months and 10 days (as shown in figure 3); no significant pest development was found after 10 months and 30 days (as shown in fig. 4).
The case shows that the artificially domesticated Autographa californica nuclear polyhedrosis virus has obvious control effect on the yellow poplar yellow silk wild borer, and compared with the traditional chemical pesticide, the artificially domesticated Autographa californica nuclear polyhedrosis virus is safe and effective for a long time.
In order to explore the cross-host infection of the virus, the inventor also conducts a large number of related experiments on indoor culture of the populus deltoids, use concentration of polyhedra, treatment of feeding amount of branches and leaves of the buxus microphylla and the like, and details are not repeated here. )
In conclusion, the nuclear polyhedrosis virus domesticated by the invention has strong control effect on the yellow poplar stem borer, the killing rate is more than 90 percent, and the nuclear polyhedrosis virus domesticated by the invention has the effects of long control time, high control efficiency and no damage to natural enemies; the invention directionally cultures the needed virus by an indoor domestication method, has strong practicability and high domestication efficiency compared with the prior field virus collection preparation and living body culture domestication, and can greatly save time, cost, manpower and material resources.
The aspects, embodiments, features and examples of the present invention should be considered as illustrative in all respects and not intended to be limiting of the invention, the scope of which is defined only by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.
The use of headings and chapters in this disclosure is not meant to limit the disclosure; each section may apply to any aspect, embodiment, or feature of the disclosure.
Throughout this specification, where a composition is described as having, containing, or comprising specific components or where a process is described as having, containing, or comprising specific process steps, it is contemplated that the composition of the present teachings also consist essentially of, or consist of, the recited components, and the process of the present teachings also consist essentially of, or consist of, the recited process steps.
Unless specifically stated otherwise, use of the terms "comprising", "including", "having" or "having" is generally to be understood as open-ended and not limiting.
It should be understood that the order of steps or the order in which particular actions are performed is not critical, so long as the teachings of the invention remain operable. Further, two or more steps or actions may be performed simultaneously.
While the invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims.
Claims (8)
1. A nuclear polyhedrosis virus domestication method suitable for yellow poplar silk wild borer is characterized by comprising the following steps:
1) providing nuclear polyhedrosis virus, adding water to dilute by 10-20 times, and applying to branches and leaves of Buxus microphylla to obtain a virus-containing feed;
2) treating the yellow poplar silk moth larvae with matrine, feeding the starved yellow poplar silk moth larvae with the virus-containing feed, collecting the virus-infected insects or dead insects, crushing, filtering, centrifuging the filtrate, collecting, and sterilizing to obtain domesticated free nuclear polyhedrosis virus;
3) taking the domesticated free nuclear polyhedrosis virus as a virus source, repeating the steps 1) to 2), and increasing domestication times;
4) and (3) carrying out insect body culture and amplification on the domesticated free nuclear polyhedrosis virus obtained in the step 3), and then purifying to finally obtain the nuclear polyhedrosis virus suitable for the yellow poplar wild borer.
2. The acclimatization method according to claim 1, characterized in that step 1) comprises: diluting the nuclear polyhedrosis virus pesticide with sterile water, and soaking branches and leaves of Buxus microphylla.
3. The acclimatization method according to claim 2, characterized in that: the volume ratio of the nuclear polyhedrosis virus pesticide to the sterile water is 1: 10 to 20.
4. The acclimatization method according to claim 1, characterized in that: the nuclear polyhedrosis virus comprises Autographa californica nuclear polyhedrosis virus, ectropis obliqua nuclear polyhedrosis virus, Spodoptera litura nuclear polyhedrosis virus or Spodoptera exigua nuclear polyhedrosis virus.
5. The acclimatization method according to claim 1, characterized in that step 2) comprises: placing eggs of the yellow poplar silk borers in a climatic incubator for incubation and colony breeding, and selecting 3-4-year-old yellow poplar silk borer larvae, wherein the culture conditions of the climatic incubator comprise: the temperature is 25.5-26.5 ℃, the humidity is 55-65%, and the ratio of the illumination time to the dark time is 14-12:10 to 12.
6. The acclimatization method according to claim 5, characterized in that step 2) comprises: starving the 3-4-year-old matrine-treated yellow poplar wild borer larvae for 12-24 hours, feeding the virus-containing feed, feeding normal branches and leaves of the small leaf yellow poplar, observing, and collecting the virus-sensitive insects or dead insects.
7. The acclimatization method according to claim 1, wherein the acclimatization generation in step 3) is 20-30 generations.
8. A nuclear polyhedrosis virus adapted to Photinus chrysosporium obtained by the acclimatization method according to any one of claims 1 to 7.
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