JPH06217766A - Production of pathogenic virus inclusion body of insect belonging to lepidoptera - Google Patents

Abstract

PURPOSE:To obtain many pathogenic virus inclusion bodies of insects belonging to the Lepidoptera in a large yield by administering a juvenile hormone(-like active substance) to larva of an insect of the Lepidoptera, breeding the larva and infecting the larva with pathogenic virus during breeding. CONSTITUTION:A juvenile hormone or a juvenile hormone-like active substance is administered to the larva of an insect of the Lepidoptera and increase of weight is continued while suppressing pupation of larva. Pathogenic virus particles are injected during breeding of larva or polygonal body or granule of pathogenic virus is taken together with feed to infect the larva with the virus and a virus inclusion body of pathogenic virus is harvested from dead larva body.

Description

Detailed Description of the Invention

[0001]

The present invention relates to inoculation of a larva of a insect of the order Lepidoptera with a pathogenic virus as a host to cause disease.
In a method for producing virus polyhedra or granules by collecting polyhedra or granules of the virus from a larva body dying of a viral disease after rearing, a juvenile hormone or a juvenile hormone-like active substance is administered to a larva By maintaining the larvae while suppressing the pupation by continuing to increase the weight of the larvae, and at the same time as the above administration, after the administration or before the administration, the larvae are infected with a viral disease and the virus propagated inside the larvae The present invention relates to a method for efficiently producing a polyhedron or a granule (generally referred to as a virus inclusion body) of the virus by harvesting from a dead larva in the form of a virus polyhedron or a granule.

[0002] The method of the present invention can be effectively used for controlling a pest carried out by artificially infecting a virus produced by this method with a pest whose host is the virus.

[0003]

2. Description of the Related Art It is known that insects of the order Lepidoptera will control pests by artificially infecting the larvae of the insects with the virus, which is a natural enemy of the insects. This pathogenic virus remains inside the cells of dead larvae as polyhedra or granules, which are a collection of virus particles encapsulated in protein crystals, and such polyhedra and granules are virus inclusion bodies. Is collectively called.

Up to now, virus inclusion bodies of phospholeptic insects such as Lotus japonicus, Spodoptera litura, Pieris rapae, Pseudococcidae, Pleurotus cornucopiae, Pleurotus cornucopiae (for example, nuclear polyhedrosis virus which is a group of baculovirus, and granulopathic virus). And virus inclusion bodies such as cell polyhedrosis virus, which is a group of reoviruses, are produced by injecting the virus into the insect larva that is the host of the virus or containing virus particles. Larvae are infected with a virus by feeding them with feed, and the larvae affected are bred, the larvae affected are bred, and the virus inclusion bodies formed in the larvae that die without pupation are collected. Is known ("Microorganisms and Agriculture", pp. 195-201,
Published by the National Association for Rural Education in 1987; "Fruit Tree Experiment Station Report", A3, pages 87-99;
"Agriculture and Horticulture," Vol. 40, No. 11, pp. 1756-17
60 pages, published by Yokendo in 1965).

For example, the production of polyhedrosis of nuclear polyhedrosis virus using Hansmon yotou as a host is carried out by infecting larvae with the nuclear polyhedrosis virus at the 5th instar (1st instar) of the larva, A method is known in which polyhedra are recovered from sick and dead larvae by sickening and dying at the age (end of age) ("Chinese Agricultural Experiment Station Report", E No. 12, pp. 46 to 62, published by the Ministry of Agriculture, Forestry and Water in 1977). When the larvae affected by the viral disease become pupae,
Recovery of viral polyhedra from the pupa is not efficient and is rarely performed.

[0006]

In view of such circumstances, the present inventors have conducted various studies on a method capable of efficiently producing virus inclusion bodies of pathogenic viruses of many Lepidoptera insects in high yield. , If a larva is raised by administering a pathogenic virus and a juvenile hormone or a juvenile hormone-like active substance (hereinafter, abbreviated as JH unless otherwise specified) to a larva of a Lepidoptera insect, the larvae are bred. It was found that pupation can be suppressed even after illness and weight gain can be continued to increase the rate of larvae that die of malady in larvae, and that the total body weight of dead larvae in which virus inclusion bodies are easily recovered can be increased. , It was found that the virus inclusion bodies from the whole larvae which died in such a manner could be efficiently recovered in high yield.

That is, the gist of the present invention is
A larva of a lepidopteran insect is administered with a juvenile hormone or a juvenile hormone-like active substance to suppress the pupation of the larva, and the larva is bred so that the weight increase of the larva is continued. Before, after, or at the same time as the administration of the hormone or the juvenile hormone-like active substance, the larvae are caused to feed on the polyhedra or granules of the pathogenic virus together with food, or
Alternatively, by injecting the virus particles, the larvae are ill, and the virus inclusion bodies of the pathogenic virus are harvested from the larvae of the larvae that have died of sickness after breeding in this manner. A high-yield production method of virus inclusion bodies of pathogenic virus.

Next, the method for producing a virus inclusion body of a Lepidoptera insect according to the present invention will be described in detail.

[0009] Examples of the Phyridales insects used in the method of the present invention include Spodoptera litura, Spodoptera litura, Pieris rapae, Spodoptera litura, Scutellaria, Chachamaki, Spodoptera litura and the like.

After sterilizing egg masses of these insects, hatched larvae are usually subjected to the aseptic rearing method, that is, temperature,
Raise the humidity so that it is suitable for co-test insects.

The period of larvae caused by the virus is from the first instar.
At the end of life, the time of the larva to give JH is 1 to 1 year before the end
Both are preferably terminal age. For example, in Hansmon yotou, the temperature is maintained at 25 to 27 ° C and the temperature is 40 to 80%, and the animal is raised and managed by artificial feed (Insector LF, etc.) until the end of its life.

The larva of the Lepidoptera insect thus obtained is inoculated with the virus obtained by the following method at an appropriate time during the breeding.

The polyhedra or granules (virus inclusion bodies) used as an inoculum can be used in any purified state, but they are bred so as not to die of other causes or to be infected with other diseases. Using larvae that died of viral disease, homogenize such larvae by grinding with deionized water, and then homogenize the homogenized solution by a conventional method 500-1.
Filter with a filter of 0 μm, 300 to 15,000 ×
Centrifugation is performed at a centrifugal acceleration of g and the purified virus inclusion body suspension may be used as an inoculum source. The centrifugal acceleration of centrifugation is preferably 300 to 4,000 for polyhedra.
It is 0 × g, and in the case of granules, it is 2,500 to 15,000 × g. For example, for Spodoptera litura, those purified by filtration through a filter of 100 to 20 μm and centrifugation at 600 to 3,000 × g may be used.

The virus is inoculated into the larvae by a method of feeding the virus polyhedron or granules (virus inclusion bodies) together with the food, or by injecting the virus particles into the larvae. For example, when a polyhedron is inoculated with Hasmontou by feeding, a suitable concentration range of the polyhedron to be mixed with the food is 0.5 × 10 ⁷ to 1 × 10 ^{6 in} terms of the number of polyhedra.
⁹ pieces / g. It is preferably 2 × 10 ⁷ to 1 × 10 ⁹ pieces / g.

The administration of JH is carried out by feeding JH together with food or by spraying a solution of JH on larvae or food. For example, in Hasmonyoto, the amount of methoprene as JH is 10 to 100 ppm.
It is preferable to feed the food contained at a concentration of 50 ppm. The dose of JH is sufficient to suppress the pupation of larvae at a high rate, and the larvae after viral disease can be bred and survived while continuing to gain weight until the end of the larval stage, but die after that. Suitable amount for.

The virus inoculation and the JH administration can be carried out at the same time or before or after, and thereafter, a temperature suitable for the co-test larvae until the larvae die of the viral disease,
It is advisable to continue breeding while maintaining weight gain under normal conditions until the end of the larvae end, while controlling the humidity.

As the JH which can be used in the method of the present invention,
Juvenile hormones JH-I and JH- described in "Biochemistry Dictionary", p. 1308 (published by Tokyo Kagaku Dojin in 1988).
II, JH-III, JH-O, and "Development of New Pesticides and Market Outlook", pages 97 to 99 (published by CMC in 1987), i.e., juvenile hormone-like active substances, methoprene, phenoxycarb, and quinoprene. , Pyriproxyfen and the like. It is preferable to use a juvenile hormone-like active substance which is considered not to be metabolized in the insect body because the effect of JH is sustained and the administration is only required once.

These JHs may be commercially available products, or they may be isolated from insect tissue or synthesized.

The sick and dead body obtained by the above-mentioned method is collected and ground with deionized water to obtain a suspension containing the virus inclusion body, which is separated and purified to obtain a virus inclusion body having infectivity. It can be collected efficiently.

Hereinafter, the present invention will be described in detail with reference to Examples, which are merely examples and do not limit the method of the present invention.

[0021]

[Example 1] An egg mass of Spodoptera litura is sterilized by immersing it in 70% ethanol for 2 minutes and further in 5% formalin for 2 minutes, and the larvae after hatching are kept at 25 to 27 ° C and a humidity of 60 to 80%, and artificial. A feed (Insector LF-manufactured by Nippon Nosan Kogyo Co., Ltd.) was given, and the animals were raised to the end of life.

Then, the larvae struck and killed by Spodoptera litura nuclear polyhedrosis were ground, and ion-exchanged water was added in an amount of about 5 times the total body weight of the dead larvae. In the example, a suspension containing the virus inclusion body of the Spodoptera litura nuclear polyhedrosis virus was referred to as "polyhedron") was filtered using a filter having a pore size of 20 μm to remove the residue. The filtrate was centrifuged at 3000 × g and the supernatant was removed to obtain a precipitate. The obtained precipitate was suspended in about 5 times the volume of the ion-exchanged water and the number of polyhedra was counted using a hemocytometer. Based on the thus obtained polyhedron concentration, 1 × 10 ⁹ polyhedron suspension was prepared by diluting with ion-exchanged water.

The polyhedron thus obtained was used as a larval inoculum.

Then, 50 ppm of methoprene and a polyhedron of a predetermined concentration were uniformly mixed with an artificial feed (Insector LF-manufactured by Japan Nippon Agricultural Industry Co., Ltd.). The artificial feed containing the polyhedron thus obtained was fed to 20 final larvae of Spodoptera litura for 2 days at 27 ° C. and 60% humidity, and then the artificial feed of the same composition containing no methoprene and polyhedron. The animals were kept for 10 days under the same temperature and humidity conditions.

As a control test A, larvae were bred from the beginning on a feed that did not contain methoprene and polyhedra (control group A), and as a control test B, polyhedra were mixed but methoprene was used. Larvae were bred with a feed that did not contain the compound (control section B).

Table 1 below shows the results of measuring the change in body weight of each of the final larvae of the larvae fed with the diet containing methoprene but containing no polyhedron over the first to tenth days of the termination period.

[0027]

[Table 1]

In the above polyhedron inoculation test, the polyhedron concentration in the feed, the number of polyhedrons that could be harvested from each dead larva, the mortality rate of larvae that died without pupation within 10 days of breeding, and the mortality The relationship between the total yield of polyhedra harvested from the whole larvae was investigated, and the test results are shown in Table 2 below.

[0029]

[Table 2]

The investigation was carried out from 4 to 10 days after the breeding by feeding the artificial feed in the larval end stage.
Each dead larva that died due to viral morbidity was ground and suspended with 5 ml of ion-exchanged water, and one drop of the resulting suspension was placed on a hemocytometer to determine the number of polyhedra per head. It was measured. The mortality rate of larvae was calculated by the following formula (1). The maximum and minimum values of the number of polyhedra per sick and dead larvae are the number of polyhedra obtained from the larvae that showed the largest polyhedron production amount among the 20 larvae tested, and the minimum number The number is shown as indicating the number of polyhedra obtained from the larvae.

When the larvae become pupae and the mortality rate of the larva is reduced, the total yield of polyhedra is reduced by that proportion. Therefore, the total yield of polyhedra was calculated by the following formula (2).

The control section A was carried out according to the method described in "Chinese Agricultural Experiment Station Report", E No. 12, pp. 46-62 (published by the Ministry of Agriculture, Water and Fisheries in 1977). 5 in Control Zone B
Since the total yield of polyhedra decreases due to age death and pupation, only 6-year-old larvae were killed in the control group.

The results are shown in Table 1.

[0034]

[0035]

EFFECTS OF THE INVENTION According to the method of the present invention, a large amount of virus inclusion bodies of pathogenic virus of Lepidoptera insect can be obtained with almost the same effort as in the prior art, and thus the virus is used as a host. It is useful for the production of natural enemy viruses used for insect control.

Claims (1)

[Claims] 1. A larva of a lepidopteran insect is administered with a juvenile hormone or a juvenile hormone-like active substance to suppress the pupation of the larva, and the larva is bred so that the larva's weight increase continues, and the larvae are reared. Before, after, or at the same time as the administration of the juvenile hormone or juvenile hormone-like active substance, the larvae are caused to feed on the polyhedra or granules of the pathogenic virus together with the food, or the virus particles are injected. The inclusion body of the pathogenic virus of the Lepidoptera insect is characterized in that the inclusion body of the pathogenic virus is harvested from the larva body of the larva sickly killed after being reared in this way. High-yield production method.