JP3524938B2 - Method for producing viral inclusion bodies of pathogenic viruses of Lepidoptera - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to inoculation of a large number of larvae of a host insect with a pathogenic virus whose original host is an insect of one species of the order Lepidoptera, to cause illness, and after the breeding. In a method for producing a virus polyhedron or granules by recovering the polyhedron or granules of the virus from a larva body killed by a viral disease, a juvenile hormone mixed in a feed given to larvae or juvenile hormone-like active substance reared larvae while suppressing pupation by administering to the larvae in the final instar larvae was continued weight gain of larvae, yet during the feed, the A polyhedron of a virus that uses an insect or its larva as its original host
The granules should be placed by mixing them in the feed at an appropriate inoculation concentration.
And thus infecting and infecting larvae with the virus ,
By harvesting the virus propagated in the reared larva from the dead larva in the form of virus polyhedra or granules,
The present invention relates to a method for efficiently producing a virus polynuclear body or a granule body (collectively referred to as a virus inclusion body) having infectivity of the virus at high yield.

[0002] The method of the present invention, the virus produced by this method, the virus may be effectively used for controlling pests performed artificially infected with pests shall be the host.

[0003]

BACKGROUND OF THE INVENTION lepidopterous is when infected with a virus is that of a natural enemy microorganism, carrying out the control of pests infected artificially virus larvae insect because Kaka illness is known. The pathogenic virus, the cells inside the worms of died larvae remain as polyhedra or granules is a collection of embedded virus particles in the crystal of a protein, such polyhedra granules viral 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. infected larvae with a virus by causing the ingested with feed, is Kaka disease, reared the Kaka disease larvae, be carried out in a manner to recover the viral inclusion bodies formed in the larval body that died without pupation 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).

[0005] For example, fifth instar larvae production of polyhedrin nuclear polyhedrosis virus lotus Mon'yotou the host (the last instar 1
Larvae infected with nuclear polyhedrosis virus
A method is known for recovering polyhedra from sick and dead larvae by sickening and dying at the 6th instar (end of age) (“China Agricultural Experiment Station Report”,
E No. 12, pp. 46-62, published by the Ministry of Agriculture and Water in 1977). When larvae disease Kaka to the viral disease is pupae, virus recovery polyhedra from pupae body it is not made less so inefficient.

[0006]

In view of such circumstances, the present inventors have made various studies on a method capable of efficiently producing virus inclusion bodies of pathogenic viruses of many species of Lepidoptera insects in high yield. I did . As a result, larvae of a Lepidoptera insect are larvae of a pathogenic virus whose host is the insect and a juvenile hormone or a juvenile hormone-like active substance (hereinafter, these are abbreviated as JH unless otherwise specified). If larvae are raised by administering them with feed at an appropriate time during the termination period of larvae, pupation is suppressed even after the viral disease , and the larvae can be raised while continuing to increase the weight of individual larvae.
Can increase the illness rate of larvae died remain larvae instar end, also the total number of larvae was found that can increase the total weight of all the number of recovered easily died larvae viral inclusions were died in this way From this, it was found that the virus inclusion body having infectivity can be efficiently recovered in a high yield.

[0007] That is, the first gist of the present invention is that the larva of a Lepidoptera insect is in the final stage of the final stage of the final stage.
In addition, by administering to the larva a juvenile hormone or juvenile hormone-like active substance that is mixed in the feed in an effective pupation-suppressing amount, the larva is controlled so that the larvae continue to gain weight while suppressing the pupation of the larva. reared, yet it feeds in to give the larvae in the breeding
, The juvenile hormone or juvenile hormone-like active substance and the co
In addition, the polyhedron or granules of a natural enemy pathogenic virus whose larvae of the Lepidopteran insect are originally unique hosts are mixed.
The polyhedra or granules are fed with the feed to cause the larva in the rear to be infected, and then the feed to be fed to the larva is fed with the juvenile hormone.
Or the juvenile hormone-like active substance is also the polyhedron or granule
Be given by switching to feed not containing, thus reared larvae continue bred while continuing weight gain until larval final instar end by suppressing pupation, further, a final instar end
A high-yield production method of a virus inclusion body having the infectivity of a pathogen virus of a Lepidoptera insect, characterized in that a virus inclusion body of the pathogenic virus is harvested from the body of a sick and dead larva.

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

[0009] Examples of the Phyridales insects used in the method of the present invention according to the first aspect of the present invention include Spodoptera litura, Spodoptera litura, Pieris rapae, Spodoptera litura, Hamaki, Chachamaki, Spodoptera litura.

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.

[0011] The timing of the larvae to be susceptible to the virus are under <br/> final instar larvae, period when Ru given the JH is in the larval instar stage, simultaneous or substantially simultaneous with the virus inoculation and JH administration <br/> For example, in Hasmonyoto, the humidity is 25 to 27 ° C.
Kept at 40% to 80%, and husbandry by artificial feed (such Insecta LF) to a final instar end.

[0012] larva of the thus-obtained lepidopterous, inoculated with the virus obtained by following the first life in the final instar larvae in 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, Hasumon path of life when inoculated by feeding the polyhedra c, the appropriate concentration range 0.5 × 10 ⁷ ~1 × 10 in polyhedra number of polyhedra mixing the bait
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 dosage of JH is an amount sufficient to suppress the pupation of larvae at a high rate, yet the virus Kaka illness of larvae rearing while continuing weight gain until the larvae final age end, can survive to illness after the Suitable amount for.

Inoculation of virus and administration of JH are
It can be carried out at the same time or at substantially the same time at the beginning of the termination period , after which it will be kept under normal temperature and humidity until the larvae die of a viral disease at the end of the termination period while controlling the temperature and humidity suitable for the co-test larvae. Under the conditions, the breeding may be continued while continuing the weight gain until the end of the larva.

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.

As a preferred embodiment when the present invention of the first aspect is applied to a specific insect, Spodoptera litura,
In the present invention of the gist of the above, in the present invention, the end of the larval
In the early stage, ie, in the 6th or 6th stage, a juvenile hormone or a juvenile hormone-like active substance mixed in the feed in an effective pupation-suppressing amount is administered to the larva to suppress the pupation of the larva. while breeding the larvae as body weight increase of larvae continues, tooth
In the feed fed to the larvae during breeding, the polyhedron or granules of the pathogenic virus of Spodoptera litura nuclear polyhedrosis with the juvenile hormone or juvenile hormone-like active substance are added to the feed at 0.5 × 10 ⁷ cells / g- Mixed at a concentration of 1 x 10 ⁹ pieces / g
By feeding the polyhedron or granules with the feed to the larva in the rear, the larva in the rear is infected, and the feed to be fed to the larva is then fed to the juvenile form.
Or juvenile hormone-like active substance
Body be given switch to feed not containing, illness common cutworm larvae were reared in this way continues to bred while continuing to weight gain until larval final instar end to suppress pupation, further, a final instar end larvae died rate of larvae is 65 to 100
%, The virus inclusion body of the pathogenic virus is harvested from the larva of the dead larva.
Provided is a high-yield production method of virus inclusion bodies having the infectivity of Hasmon Yoto, a nuclear polyhedrosis virus. The method of the present invention according to the second aspect can be carried out in the same manner as the method of the first present invention. 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 concentration of polyhedra incorporated in the feed, the number of polyhedra that could be harvested from each dead larva, the mortality rate of larvae that died without pupation within 10 days of rearing, 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 B was performed according to the method described in "China Agricultural Experiment Station Report", E No. 12, pages 46 to 62 (published by the Ministry of Agriculture, Forestry and Fisheries in 1977). In control section B, the total yield of polynuclear bodies decreases due to the death of the 5th instar and pupation,
The number of sick and dead larvae in the control group was only 6th-instar larvae.

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.

─────────────────────────────────────────────────── ───Continuation of front page (56) Reference JP-A-3-236747 (JP, A) JP-A-1-132324 (JP, A) Journal of the Kansas Entomological Society, 1981, Vol. 54, No. 54 3, p. 489-495 China Agricultural Experiment Station Report E, 1977, No. 12, p. 46-52 (58) Fields surveyed (Int.Cl. ⁷ , DB name) C12N 7/00

Claims (2)

(57) [Claims] 1. The end stage of the larvae of the insect Lepidoptera
In the early stage of the administration, juvenile hormone or juvenile hormone-like active substance mixed in the feed in an effective amount to suppress pupation is administered to the larva so that the larvae continue to gain weight while suppressing pupation of the larva. bred larvae, yet given to the larvae in the breeding
In addition to juvenile hormones or juvenile hormone-like active substances , polyhedra or granules of pathogenic viruses of natural enemies whose larvae of the Lepidopteran insects are their original hosts are mixed in the feed.
It is included in the polyhedron with the feed or
Feeding granules to live larvae causes the live larvae to become sick, and then feed the larvae to young larvae.
Hormones or juvenile hormone-like active substances
Giving switched to the feed containing neither granules is such bred larvae continue bred while continuing weight gain until larval final instar end by suppressing pupation, further, final decree
A high-yield production method of a virus inclusion body having the infectivity of a pathogen virus of a Lepidoptera insect, which comprises harvesting a virus inclusion body of the pathogenic virus from the body of a dead larva that died at the end of the term . 2. The final stage of the larvae of Spodoptera litura, that is,
In the early stage of the 6th stage, the juvenile hormone or juvenile hormone-like active substance mixed in the feed in an effective amount to suppress the pupation is administered to the larva to suppress the pupation of the larva while suppressing the pupation of the larva. reared larvae as weight gain continues, yet is in the feed given to the larvae in the breeding, juvenile hormone or juvenile
0.5 x 1 of the polyhedra or granules of the pathogenic virus of Spodoptera litura, along with hormone-like active substances, in the feed
It is mixed at a concentration of 0 ⁷ pieces / g to 1 × 10 ⁹ pieces / g.
There, is diseased larvae in rearing by feeding larvae in rearing the multi angle, or granules with feed, its
, Feed the larvae with a juvenile hormone or juvenile
Does not contain hormone-like active substances or polynuclear bodies or granules
There is given switches to feed, Spodoptera litura larvae were reared in this way continues to bred while continuing weight gain was suppressed pupate until larval final instar end, further, a final age end
After the larval mortality rate of larvae dying of the larval stage reaches 65 to 100%, the virus inclusion bodies of the pathogenic virus are harvested from the larvae of the larvae of the illness. A high-yield production method of a virus inclusion body having infectivity.