JP2010183902A - Method for creating ladybug genetically defective in flying ability - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for creating a ladybug genetically defective in flying ability that can maintain pest insect control effect without reducing reproductive ability and the survival rate of larva depending on advance of generation. <P>SOLUTION: The method is to create a ladybug genetically defective in flying ability and comprises a process for creating a ladybug lineage genetically defective in flying ability by selecting individual ladybugs having low flying ability out of an arbitrary ladybug group depending on flying distances and repeating operations to obtain the next generation through mass mating of the selected individuals, and a process for inter-lineage mating of the created lineages themselves or a process for mating a ladybug genetically defective in flying ability with an individual just after collected from outdoors, and a process for backcrossing the obtained first generation hybrid (F<SB>1</SB>) with the parent ladybug lineage defective in flying ability. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing a ladybird lacking genetically flying ability useful as a natural enemy preparation for aphids.

  Aphids are known as a hard-to-control insect in many crops, fruit trees, and vegetables. Until now, aphids have been mainly controlled by chemically synthesized insecticides, but problems such as the effects of insecticides on the ecosystem and human body and the development of pesticide resistance have occurred. There is a strong demand for the development of biocontrol technology that mainly uses In Japan, four kinds of natural enemy preparations have been put to practical use for aphids, such as Koreman abachi, Shobata flies, Yamasokusakaerou, and ladybird. However, since the environment in which existing natural enemy preparations can be used is limited to institutional cultivation conditions, chemical control is mainly carried out mainly by chemical synthetic insecticide spraying in outdoor cultivation conditions.

  Since adults of Nami Tentou are predatory natural enemies of aphids, they have already been used as natural enemy preparations as described above. However, there is a problem that the rate of fixation in a greenhouse is low because of high flying dispersion ability. In order to cope with this problem, adult Nami ladybugs that have become unable to fly by physically folding the cocoons are sold as biological pesticides in Japan (Non-patent Document 1), but the production cost of ladybirds until they become adults Very high (Non-Patent Document 2). On the other hand, if Nami ladybug flight is disabled, it can be released not only in adults, but also in the developmental stage of eggs and larvae. Reduction can be expected. In France, Nami Tento is genetically lacking flight ability (Patent Document 1), and its line is sold in Europe. In Japan, it has been studied to produce Nami Tento lacking the ability to fly genetically based on a group collected outdoors (Non-patent Document 3). Nami Tento lacking genetically flying ability is highly established even in outdoor cultivation conditions, and it is expected to be used in many outdoor cultivation conditions for vegetables and flower buds that had previously had to rely on chemical control. The However, this genetically non-flying Nami tentou has a high rate of establishment of the released Nami tentou, but the number of next-generation individuals is small. Therefore, it was necessary to release a lot of Nami Tento in order to sufficiently suppress the occurrence of aphids.

  On the other hand, when natural enemy insects are bred for a long time under artificial breeding conditions, the genetic composition of the strain may change due to effects such as adaptation to the breeding environment, genetic drift, and inbreeding (Non-Patent Document 4). In particular, in the line of natural enemy insects created by repeated selection and similar mating in order to give characteristics effective for pest control, inbreeding is proceeding considerably, and inbreeding is likely to occur. There is a risk that the survival rate is lowered, that is, the pest control effect by the next generation of the released individuals is reduced. In Namitentou, which lacks the ability to fly genetically, egg hatching rate, larval survival rate, and number of adult laying eggs have decreased with the progress of generations, and these effects are reflected in the decrease in aphid control effect. (Non-Patent Document 5).

International Publication No.98 / 24308

“New Natural Enemy Pesticides: How to Use Nami Tento Agents”, Toshiyuki Tezuka, 2003, Plant Protection, 57, 376-379. `` Natural enemy ecology and utilization technology '', Eiji Yano, 2003, Yokendo, p.59-60. Seko, T., Yamashita K., Miura, K. (2008) Residence period of a flightless strain of the ladybird beetle Harmonia axyridis Pallas (Coleoptera: Coccinellidae) in open fields, Biological Control, 47, 194-198. Hopper, K. R., Roush, R. T., & Powell, W. (1993) Management of genetics of biological-control introductions. Annual Review of Entomology, 38, 27-51. `` Effects and countermeasures of long-term selection on survival and reproduction in non-flying Nami Tentou, and countermeasures '', Tomoichi Seko and Ichiki Miura, 2008, 13th Agricultural and Forestry Pest Control Study Group Report, p.31.

  It is an object of the present invention to provide a method for producing a ladybird that lacks the ability to fly genetically and can maintain a pest control effect without reducing the reproduction ability and the larval survival rate due to the progress of generations.

As a result of intensive research to solve the above problems, the present inventors have created a plurality of Nami Tento lines that genetically lack flight ability, and when different lines are crossed, the reproduction ability and the larval survival rate are restored. It was found that Nami tentou obtained by crossing between the strains exhibited excellent next-generation effects. In addition, by crossing the Nami Tento line genetically lacking flying ability with an individual immediately after being collected from the field, backcrossing the Nami Tento line with the parent's genetic lacking flying ability back to the hybrid first generation (F ₁ ) Also found that the same effects as cross-crossing can be obtained. The present invention has been completed based on such findings.

That is, the present invention includes the following inventions.
[1] A method for producing a ladybird genetically lacking flight ability, including the following steps.
(1) A process of selecting a ladybird having a low flying ability from an arbitrary ladybird group, repeating the operation of obtaining the next generation by grouping the selected individuals and obtaining a ladybird group having a low flying ability
(2) From the population obtained in (1), one male and one male are mated and paired, and the pair with the lowest flight ability of the next generation individual is selected as a ladybird lineage lacking flight ability. The process of creating a ladybird strain A that genetically lacks flight ability by further repeating population mating
(3) A process for producing a ladybird line B genetically lacking flight ability by performing the same operations as in (1) and (2) above in another ladybird population
(4) Step of crossing the ladybird line A and the ladybird line B
[2] The method according to [1], wherein the operation of obtaining the next generation by group-mating selected ladybirds with low flying ability is performed for 20 generations or more.
[3] A method for producing a ladybird genetically lacking flight ability, including the following steps.
(a) A step of selecting a ladybird having a low flying ability from an arbitrary ladybird population, repeating the operation of obtaining the next generation by group-mating the selected individuals and creating a ladybird strain C genetically lacking the flying ability
(b) crossing the ladybird line C with a ladybird group (wild line) collected from the field, and backcrossing the ladybird line C to the obtained hybrid (F ₁ )
[4] The method according to any one of [1] to [3], wherein the ladybird is a ladybird that prey on aphids.
[5] The method according to [4], wherein the ladybirds that prey on the aphids are Nami ladybirds ( Harmonia axyridis ), Nana hoshi ladybirds ( Coccinella septempunctata ), Dandara ladybugs ( Menocchilus sexmaculatus ), or Propylaea japonica .
[6] A ladybird strain genetically lacking flight ability obtained by the method according to any one of [1] to [5].
[7] A biological pesticide comprising, as a main component, a ladybird strain genetically lacking flight ability obtained by the method according to any one of [1] to [5].

  According to the present invention, a ladybird line genetically lacking in flight ability is created by the intercrossing or backcrossing technique. A genetically lacking flying ability Nami Tento line produced by the present invention exhibits excellent next-generation effects without losing the “does not fly” property imparted by selection. Therefore, even if it is released in a small amount, a sufficient effect of controlling aphids is exhibited, and it is also possible to release at the egg or larva stage. Therefore, this ladybird strain can be put into practical use as a biopesticide that can stably control aphids at low cost, and can contribute to a significant reduction in the use of insecticides in the cultivation of vegetables and flowers on the ground as well as facilities.

The schematic diagram of the hybrid system | strain production by the cross | bridging between the strain | stump | stock (line | system | group A, system | strain B) of two genetically lacking flight ability obtained by artificial selection is shown. It is the figure which showed that the survival rate and reproductive capability of a hybrid system | strain were excellent. The left figure shows the egg hatching rate of line A, line B and their hybrid lines. The numbers are the number of egg masses measured, and there is a significant difference between the data with different English letters (Tukey- Kramer test, p <0.05). The middle figure shows the emergence rate of line A, line B, and their hybrid lines, that is, the survival rate in the larval stage. The numbers in the figure are the number of larvae used in the measurement, and are significant among the data with different English letters. There is a difference (Sequential Bonferroni method, p <0.05). The right figure shows the number of eggs laid in line A, line B and their hybrid lines. The numbers are the number of females used in the measurement, and there is a significant difference between the data with different English letters (Tukey-Kramer test, p < 0.05). It shows the development and development of the number of aphids per eggplant leaf in strains A, B, greenhouses that released adults of these hybrid strains, and untreated greenhouses. The arrows indicate that adults of Namitenta were released at a density of 1 per eggplant strain. The number of Namitentou 4th instar (final) larvae generated in a greenhouse that has released adults of line A, line B, and hybrid lines thereof, that is, the developmental fate of the next-generation individuals of the released adults is shown. The schematic diagram of the hybrid system | strain production by the backcross of the Namitentou system | strain C which lacks a flight ability genetically with an outdoor system | strain is shown. Parent generation (Namitentou line C genetically lacking flight ability with the field strain), F ₁ generation (hybrid first generation (F ₁ ) obtained by crossing the parent generation individuals), F ₂ generation (F ₁ generation individuals It is the figure which compared the flying ability of the Nami Tentou individual of the generation) obtained by backcrossing the Nami Tento that genetically lacks flying ability of the parent generation.

The present invention is a method for producing a ladybird genetically lacking flight ability, and includes the following steps.
(1) A process of selecting a ladybird having a low flying ability from an arbitrary ladybird group, repeating the operation of obtaining the next generation by grouping the selected individuals and obtaining a ladybird group having a low flying ability
(2) From the population obtained in (1), one male and one male are mated and paired, and the pair with the lowest flight ability of the next generation individual is selected as a ladybird lineage lacking flight ability. The process of creating a ladybird strain A that genetically lacks flight ability by further repeating population mating
(3) A process for producing a ladybird line B genetically lacking flight ability by performing the same operations as in (1) and (2) above in another ladybird population
(4) Step of mating the ladybird strain A and the ladybird strain B in the present invention is not particularly limited as long as the ladybird is a ladybird that prey on the aphids. For example, Harmonia axyridis , Coccinella septempunctata ), Dandara Tento ( Menochilus sexmaculatus ), or Himekame no Tento ( Propylaea japonica ).

Hereafter, each said process is demonstrated in detail.
First, in step (1), a ladybird individual having a low flying ability is selected from an arbitrary ladybird population. Here, the ladybird population is preferably a population containing about 50 to 100 males and females. About the ladybird group, the flight distance is measured using a flight measuring device flight mill, and about 30% of the ladybird individuals having a low flight distance are selected from the group. The selected individuals are mated to obtain the next generation, and the flight distance is measured in the same manner for the next-generation ladybird population, and the ladybird individuals with low flying ability are further selected. Such an operation is performed for 20 generations or more, preferably 25 to 30 generations, to obtain a ladybird population with low flying ability.

  Next, in step (2), one male and one female from the population obtained in step (1) are mated and paired, and the flight distance is measured in the same manner for the next generation individuals, with the lowest flight ability. The pair is selected as a ladybird lineage lacking flight ability. The number of pairs to be mated depends on the number of ladybirds included in the ladybird population obtained in step (1), but 10 to 15 pairs are preferred. For the ladybird lineage lacking the flying ability selected here, the mating is repeated for 3 to 5 generations within the line, and a ladybird line A genetically lacking the flying ability is created.

  Further, in step (3), the same process as in the above (1) and (2) is performed with different ladybird populations to create a ladybird line B genetically lacking flight ability. The production of the ladybird line B from the different ladybird populations in step (3) may be performed in parallel with the production of the ladybird line A, or at different times.

  Finally, in the step (4), the ladybird line A and the ladybird line B are intercrossed to obtain a hybrid line that is an object of the present invention. This hybrid line, and the ladybird line A and ladybird line B to create it are preserved in the Biopesticide Development Laboratory, a facility of the Kinki Chugoku Shikoku Agricultural Research Center (Fukuyama City). Is ready for sale.

In addition, the method for producing a ladybird that is genetically lacking flight ability according to the present invention is a method for producing a ladybird that is genetically lacking flight ability and that is produced by repeating selection and population mating similar to the above step (1) in an arbitrary ladybird population. It can also be carried out by crossing a line (line C) with a ladybird population (wild line) collected in the field and backcrossing the line C to the obtained primary hybrid (F ₁ ). Further, the above-mentioned line C may be back-crossed again to a cross individual (F ₂ ) by back-crossing.

Therefore, according to the present invention, there is also provided a method for producing a ladybird genetically lacking flight ability, including the following steps.
(a) A process of selecting a ladybird having a low flying ability from an arbitrary ladybird population, repeating the operation of obtaining the next generation by cross-breeding the selected individuals and creating a ladybird strain C genetically lacking the flying ability
(b) crossing the ladybird line C with a ladybird group (wild line) collected from the field, and backcrossing the ladybird line C to the obtained hybrid (F1)

  ADVANTAGE OF THE INVENTION According to this invention, the ladybird strain | stump | stock (hybrid system | strain) which genetically lacked the flying ability produced by one of the said methods is provided. In order to stably supply and maintain this hybrid line, artificial baits (eg, Frozen Eggs) or leaves of plants infested with aphids, egg-laying substrates (eg, tissue paper) Can be used. Specifically, about 50 male and female adults of the ladybird strain (hybrid strain) are released into this plastic container, and arbitrarily mated, so that the female adult lays eggs in the container and the next generation grows. At this time, the temperature is preferably 20 to 25 ° C. and the humidity is preferably 50 to 70%. Under the conditions described above, one generation is repeatedly grown in about 50 days. Those collected sequentially are used as biological pesticides.

  Therefore, according to the present invention, there is also provided a biopesticide comprising a ladybird line (hybrid line) genetically lacking flight ability produced by the above method as a main component. As used herein, “biological pesticide” refers to a product prepared by making the organism used for biological control easy to use as a pesticide.

  In the biopesticide of the present invention, the development stage of the ladybird strain is not limited, and it may be not only adult but also larva and egg. When adults or larvae are used, the effect of controlling aphids is excellent. On the other hand, since the control effect appears for the first time after hatching, there is no immediate effect, but it is preferably used for the purpose of preventive control. Moreover, if an egg and an adult and / or a larva are used together, the aphid control effect can be achieved in addition to the rapid effect. Therefore, since the timing of aphid control can be arbitrarily controlled by any one of eggs, larvae and adults, or a combination thereof, more effective control can be performed.

  In addition, the biopesticide of the present invention contains a ladybird strain (hybrid strain) genetically lacking flight ability produced by the above method as a main component, and other components that do not affect the survival and / or growth of the ladybird. May be. Examples of such other components include sawdust, rice bran, bran, paper pieces, fibers, vermiculite, bentonite, and the like, and serve as a bulking agent, a heat retaining agent, a moisturizing agent, an impact absorbing agent, and the like.

  As the dosage form of the biopesticide of the present invention, adults, larvae, bottles and other containers containing eggs, and paper pieces to which eggs or cocoons are attached are suitable for both distribution and use. In use, for example, an excellent control effect against aphids can be obtained by an extremely simple method of installing the bottle in a facility or outdoor area that requires control. The number of ladybirds accommodated per bottle is, for example, about 50 to 100.

Control of aphids using the biopesticides of the present invention is performed by releasing the above ladybird line (hybrid line) at the place (facility) where aphids are generated. The individual density of ladybirds necessary for control can be appropriately adjusted according to the size of the aphid generation site (facility), the degree of aphid generation, the season, and the growth stage of the ladybird used. For example, when adults are used, the density is 0.5 to 5 heads / m ² , preferably 3 to 4 heads / m ² . The “facility” is intended to encompass both inside and outside the facility.

  The time of application may be any time as long as the aphids to be controlled live, but it is preferably from immediately before the start of breeding to the initial stage. These methods are known to those skilled in the art.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these.

(Example 1) Creation of the next generation (hybrid strain) of Namitentu strains genetically lacking flight ability Using a flight measurement device flight mill, the flight distance of 50 Namitentu adults was measured, and the flight distance of The operation of selecting 30% of short individuals and obtaining the next generation was repeated. The flying distance of adults decreased with the passage of generations, and it was confirmed that 70% of adults could not fly in the 20th generation. From the 24th generation onwards, there were also generations with increased flight ability, probably because the effect on the selection had diminished, so in the 27th selection the adults of 7 males and 1 females were mated and paired together. Individuals were obtained, and the pair with the lowest flight ability of the next generation individuals was created as Nami Tento Line A, which lacks the ability to fly genetically, and group selection was continued within that line after the 28th generation. In addition, by the same method as described above, a Nami Tento line B that genetically lacks flight ability was created separately, and group selection was continued within that line. In the 8th generation and 9th generation of the line B, the number of adults of Namitenta was small, so flight measurement was not performed.

  The next generation of the desired ladybird strain that lacks the ability to fly genetically by mating 20 adult females of the 32nd generation selected from the line A with 20 male adults of the 38th generation selected from the line B. ) Was obtained (FIG. 1).

(Example 2) Evaluation of the breeding ability of the hybrid strain The egg hatching rate, the larval survival rate, and the number of adult eggs laid (for 10 days from the start of egg laying) of the hybrid strain obtained in Example 1 were examined. Both A and B were significantly improved, reaching almost the same level as the Nami Tento line (Control) with normal flight capability (Fig. 2).

(Example 3) Evaluation of aphid control effect of hybrid line When adults of hybrid line obtained in Example 1 were released in a plastic house in which eggplants were planted, line A and line B were released including the untreated section. The occurrence of aphids was significantly suppressed compared to the treated group (FIG. 3). In addition, the next generation larvae of the adult Nami Amanita were released most in the treated area where the hybrid line was released (FIG. 4).

  Table 1 shows the results of examining the flight distance per hour for the system A, the system B, and the hybrid system.

  As shown in Table 1, there was no significant difference in flight distance between line A, line B, and the hybrid line, and it was confirmed that flight ability was not recovered by crossing between lines. Among the hybrid lines, individuals with high flying ability were also observed, but only 1 to 2 animals were flying, and the flying distance per hour of other individuals was 0 m.

(Example 4) Effect of backcrossing genetically lacking flying ability with Nami Tento lines and Nami Tento individuals collected from the field About 150 Nami Tento adults were collected from the field, and the next generation adults from the group were 50 males and 50 females. Obtained (wild strain). On the other hand, in any Nami Tento group, the selection by the flight distance and the group mating of the selected individuals were repeated in the same manner as in Example 1, and the Nami Tento strain C (male and female each) that lacks the ability to fly genetically in the 40th generation from the start of the selection 50) each, and the wild-type individuals were mated together (parent generation mating). As in the case of the parent generation, these cross individuals (F ₁ generation) were cross-bred together with the 41st generation of line C, and individuals with low flying ability were selected in the F ₂ generation, which is a separate generation (FIG. 5). .

While there is no difference in the frequency of flight ability in the parent generation and F ₁ generation, the F ₂ generation flying distance appeared many individuals 0 m (Fig. 6). By performing backcrossing, F ₁ combination of male and female in two situations generation _{(F 1 -1, F 1 -2} ), F 2 male and female four combinations in generation _{(F 2 -1, F 2 -2} , F ₂ -3, F ₂ -4) occurred. In any of the combinations in the F ₂ generation, approximately half of individuals had disappeared the flight ability.

In the F ₃ generation, the flight ability of almost all individuals measured is lost, while, it was confirmed with high fertility (Table 2). The adult female of F ₃ -1 was obtained by artificially selecting individuals having a flight distance of 0 m from the F ₂ -1 strain and performing group mating. Similarly, the F ₃ -2 female adult is derived from the F ₂ -2 strain described above.

Claims (7)

A method for producing a ladybird genetically lacking flight ability, including the following steps.
(1) A process of selecting a ladybird having a low flying ability from an arbitrary ladybird group, repeating the operation of obtaining the next generation by grouping the selected individuals and obtaining a ladybird group having a low flying ability
(2) From the population obtained in (1), one male and one male are mated and paired, and the pair with the lowest flight ability of the next generation individual is selected as a ladybird lineage lacking flight ability. The process of creating a ladybird strain A that genetically lacks flight ability by further repeating population mating
(3) A process for producing a ladybird line B genetically lacking flight ability by performing the same operations as in (1) and (2) above in another ladybird population
(4) Step of crossing the ladybird line A and the ladybird line B   The method according to claim 1, wherein the operation of obtaining the next generation by performing group mating of the selected ladybird individuals with low flying ability is performed for 20 generations or more. A method for producing a ladybird genetically lacking flight ability, including the following steps.
(a) A process of selecting a ladybird having a low flying ability from an arbitrary ladybird population, repeating the operation of obtaining the next generation by cross-breeding the selected individuals and creating a ladybird strain C genetically lacking the flying ability
(b) crossing the ladybird line C with a ladybird group (wild line) collected from the field, and backcrossing the ladybird line C to the obtained hybrid (F ₁ )   The method according to claim 1, wherein the ladybird is a ladybird that prey on aphids. The method according to claim 4, wherein the ladybird that prey on the aphids is Harmonia axyridis , Coccinella septempunctata , Dendara ladybug ( Menochilus sexmaculatus ), or Propylaea japonica .   A ladybird strain genetically lacking flight ability obtained by the method according to any one of claims 1 to 5.   A biological pesticide comprising, as a main component, a ladybird strain genetically lacking flight ability obtained by the method according to any one of claims 1 to 5.

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