JPH0633241B2 - Biological control of insects with insect parasitic nematodes - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to an insect parasitic nematode detected from soil in Japan.
The present invention relates to a method of using Heterorhabditis spp. OJ-N2 as an insecticide.

[Conventional technology]

Steinernema spp. And Heterorhabdi, which are insect parasitic nematodes
When tis spp. invades an insect body, it has a common characteristic insecticidal property that the action of symbiotic bacteria causes sepsis in the body and causes it to die.

Therefore, the range of insecticidal effect is extremely wide, and its use for biological control has been studied for a long time ("Genetics" Vol. 37 No. 6 (1983) pp. 31-35. "Plant epidemics" No. 38, No. 3, (1984), pp. 31-35).
From the research results, it is found that using such nematodes as an insecticide is an extremely effective method.

However, it is said that the nematode's insecticidal power and viability are the key to the success or failure of these nematodes' pesticides.

In fact, S. glaseri, detected in the beetle larvae in the eastern United States in 1929, has a spectacular history of extensive field application for the control of beetle larvae in the 1930s and 1940s. But initially it was big
The insecticidal effect of S.glaseri also declined before long, and the interest was also weakened.

In this way, the decreased insecticidal effect of S. sglaseri on the beetle larvae was attributed to the nematode culture conditions, that is, the culture under conditions that inhibit the growth of symbiotic bacteria. It has been pointed out (Steiner, G.
(1929), J.Wash.Acad.Sci., 19: 436-440, G.aser, RW (193
1), Sci., 73: 614-615, Glaser, RW (1932), New Jersey D
epy.Agric.Circ., 211: 3-34, Glaser, RW (1940), Proc.S
oc.Exp.Biol.Med., 43: 512-514, Poinar, GOJr. (1979),
CRC.Press Inc., 227p.). In recent years, as the dangers and limits of chemical pesticides have become apparent in Japan, expectations for biological pesticides that use natural enemies existing in natural ecosystems have increased, and insect parasite nematodes have attracted attention again. Has started to collect. Under such circumstances, a group of several researchers confirmed that nematodes introduced from abroad were effective against the difficult-to-control pests in Japan even in the laboratory, but in field tests. It has been clarified that the distribution is not uniform (MEXT Science Research Grant No. 5
9860005. "Development of control methods using insect parasitic nematodes").

This is because the applied nematodes are vulnerable to dryness, ultraviolet rays, and high temperatures in the aboveground part, and mites in the underground part.
This is because there is a problem of persistence of viability due to predation by other kinds of nematodes or parasitism.

As described above, there are various problems to be solved in order to use the above-mentioned conventional insect-parasitic nematodes as biological control means. Therefore, in order to control pests in Japan, nematodes that are simultaneously present in natural ecosystems as natural enemies of pests must be used.

In other words, since the insect-parasitic nematodes that have been used in Japan so far have been introduced from abroad, they cannot adapt to the climate and ecosystem of Japan, and before they exert their insecticidal effect. There was a drawback that it would die. In order to solve such a drawback, the present inventors have conducted a study on a method of searching among the native nematodes that inhabit Japan and showing the insecticidal activity, and using this as an insecticide. As a result, the present invention has been completed.

[Problems to be solved by the invention]

The present invention relates to a method of using an insect parasitic nematode that lives in Japan and has a high insecticidal effect that is sufficiently compatible with Japan's climate and ecosystem as a biological control means (insecticide).

[Means for solving problems]

The present invention is an insect-parasitic nematode that has had problems such as a decrease in insecticidal effect because it has been introduced from abroad, the climate climate of Japan sufficiently to live in Japan, high insecticidal effect compatible with the ecosystem. The present invention relates to a method for searching for an insect parasitic nematode that has a bacterium and using it as a biological control means (insecticide).

Hereinafter, the insect parasitic nematode used in the present invention will be described in detail.

Insect parasitic nematode The nematode Heterorhabditis spp OJ-N2 (hereinafter simply referred to as OJ-N2) used in the present invention was detected from soil collected from a nursery in Hamakita City, Shizuoka Prefecture in May 1986. As a detection method, several kinds of nematodes separated from the collected soil by the Balemann method were inoculated into a Petri dish lined with paper. The larvae of Spodoptera litura are released here to infect nematodes, and the larvae that die after 2 to 5 days are taken out and placed under a constant temperature condition of 25 ° C for about 2 weeks. Recovered.

The infectious larva of OJ-N2 has a morphology as shown in FIG.

The nematode (OJ-N2) detected by the above-mentioned method infected and killed many insects such as the Spodoptera litura larva and the scarab beetle larva. The life history of this nematode OJ-N2 in the insect body and the death history of the insect were in agreement with the known species of Heterorhabditis spp. OJ invading from the mouth and anus of the larva
-Infectious larvae of N2 enter the body cavity through the intestinal tract. Here, symbiotic bacteria (Xe-norhabdus lumines) released from nematodes
cens) causes sepsis in the insects and causes the insects to die as their proliferation.

Infectious larvae grow and appear only in the first-generation adult (female adult) with the form shown in Fig. 2. This is Heterorhabditi.
s spp. ), And begins to grow in dead worms. Fig. 3 shows an enlarged view of the tail of an adult female (10 × 20
(× 1.5)). The propagated nematodes become infectious larvae again, and some of them grow to become second-generation adults. At this time, for the first time, an individual having male and female reproductive organs appears.
The second generation male has the form shown in FIG.
It has 9 pairs of sexual projections as seen on either side of the interlace as shown. In this way, a large number of nematodes are filled in the body of the dead worm, and in the moistened state, infectious larvae start to migrate out of the body.

As mentioned above, this nematode OJ-N2 was identified as Heterorhabditis spp. From the morphological characteristics and ecological characteristics.

In addition, from the same area where the main nematode OJ-N2 was separated, 19
In 1984, Kushida et al. (The 37th Nichibayashi Kanto Scholar, 163-16)
4, 1986) from Stefanernema sp.
p. is detected, but the nematode OJ-N2 by the present inventors
Is a Hete-rorhabditis spp., So both nematodes are different species.

Insecticidal test The nematode OJ-N2 infected larvae such as Lotus japonicus, Kaburagaiga, Douganebu buoy, Himekogane and the like at a higher rate than conventional insect parasitic nematodes and killed them.

Conventional nematodes show relatively insecticidal activity in the laboratory against the above-mentioned harmful insects, but often do not show insecticidal activity under natural conditions in the field. However, this nematode OJ-N
2 showed a high rate of insecticidal activity not only in the laboratory but also under natural conditions in the field. Furthermore, after propagating in dead worms under the same conditions, the migrating nematodes naturally infected the following insects and killed them.

Viability test This nematode OJ-N2 has higher viability than conventional foreign-produced nematodes because it was detected in soil under natural conditions in Japan. In fact, when the nematode OJ-N2 is applied to the soil,
It was found that the effect was maintained for a long time. Not to mention the insecticidal activity, this nematode OJ-N2 is sufficiently compatible with conventional synthetic pesticides from the viewpoint of maintaining its effect.

For example, by applying a large amount or regularly of the nematode OJ-N2 to the soil and allowing it to settle, it is possible to reduce the application amount and the number of times of application of the pesticide, and
It is possible to control the density semi-permanently below the EIL (damage allowance density) without causing resistance to pests.

Large-scale culture method As a conventional large-scale culture method for insect parasitic nematodes, a multiplication method using Lepidoptera larvae (worm body multiplication method), a dog food medium method,
Cow. Pig. A large amount of nematode infectious larvae can be easily proliferated by the chicken gut medium method in which the internal organs of a bird are soaked and used as a “use” (“Plant Protection”, No. 44).
~ P. 49. JP-A-52-41225). In addition, a pupal flour medium invented by the present inventors (Japanese Patent Application No. 62-414).
The method using 11) is an effective mass proliferation method.

Among the above four methods, the chicken gut medium method is currently put into practical use, but the nematode OJ-N2 can also basically be propagated by these four methods. Particularly, in the chicken gut medium method,
About 2 to 10 times more infectious larvae than conventional nematodes can grow at a high rate. In addition, this culturing method goes through the process of subculture, and after surface sterilization of infectious larvae that have proliferated in dead insect bodies, the above-mentioned livestock's internal organs are made into juice and sprinkled onto a medium that has been soaked in a sponge. Aseptically inoculate, 20-30
Incubate at 40 ° C for 20-40 days. The nematodes grown at this point are small and impractical. However, the number of proliferating nematodes gradually increases by inoculating the nematodes obtained here again in a new medium by the same method and subculturing several times. The nematodes that proliferated in large quantities in this way did not lose their insecticidal activity against the above-mentioned insects even after being passaged for about half a year.

Hereinafter, the present invention will be described in more detail with reference to Examples.
The invention is not limited to these examples.

Example 1 Insecticidal activity of the present nematode OJ-N2 against various insects, and comparison with conventional nematodes Test nematode OJ-N2 used infectious larvae proliferated in dead larvae of Lartus japonicus larvae . As a control, Steinernema feltiae (str.DD) grown on the above chicken gut medium was used.
-136), S.glas-eri, H. heliothidis infectious larvae were used.

Insects to be tested Three successive generations of Spodoptera litura, last-instar larvae of Aedes aegypti, and Spodoptera litura var.
Instar (white) larvae were used.

Nematode inoculation and investigation method Each insect is inoculated with the number of heads shown in Table-1 and placed at a constant temperature of 25 ° C to examine the mortality of the insects.

As a result, it was found that the nematode OJ-N2 has a high insecticidal activity about 2 to 50 times that of the conventional nematode, and is particularly effective against Coleoptera insect pests such as Himekogane and Douganebububui. .

Example 2 Comparison of the viability of the main nematode OJ-N2 and conventional nematodes mixed in soil The test nematodes OJ-N2, S.glaseri, and H.heliothidis infectious larvae were respectively treated in Example 1 The cells were proliferated by the above method and then tested.

Test soil Normal nursery soil (black soil, water content 53.3% weight ratio)
Was not sterilized and was used as it was.

Method for investigating nematode contamination and viability 500,000 nematodes were mixed into 2 kg of soil in a container (30 x 25 x 10 cm) at room temperature (15-25 ° C)
I kept it. Regarding the viability of nematodes, a part of the soil is taken out from the container, placed in a polyethylene cup, and the second instar larvae of Rhododendron buoy is released and kept at 25 ° C., and the results of its insecticidal activity are shown in Table 2.

As a result, the viability of the conventional nematodes rapidly declined immediately after being mixed in the soil, and did not show at all 15 or 30 days after mixing. This indicates that conventional nematodes show effects in the laboratory, but the effects when applied under natural conditions are not uniform. However, the effect of the present nematode OJ-N2 was maintained at a high rate even after 30 days.

Example 3 Mass Proliferation on Chicken Gut Medium Test nematode The present nematode OJ-N2 was obtained by substituting infectious larvae that proliferated in dead larvae several times on a sponge medium as in Example 1. The infected larvae were tested. Also, as a control, S.felt
iae (str.DD-136). S.glaseri and H.heliothidis were tested in the same manner as those obtained on chicken gut medium.

Preparation of Medium Fresh chicken (broiler) intestines that had been frozen and stored at −30 ° C. were thawed and tested after washing and removing the contents. Polyurethane sponge 20 into which 1 m of intestine is added with 250 m of warm water (about 50 ° C.) and ground with a household mixer for 1 minute to make it into a juse-like shape, and cut into 1 cm or less pieces
Absorbed to 0 g. 15 g of each of these media was placed in a test tube with a diameter of 3 cm and a length of 18 cm, and then autoclaved (1
20 ° C., 20 minutes).

Nematode inoculation and investigation method Infected larvae preliminarily washed 5 times with rumarin solution were surface sterilized with 0.1% merthiolate for 3 hours, and then washed 3 times with sterilized water. After 1,000 heads were inoculated into the medium using a micropipette, the cells were cultured at 25 ° C. After culturing for a certain number of days, the cells were separated by the Bertmann method and counted by a Peter 1 m counter. The insecticidal activity and viability of infectious larvae of OJ-N2 obtained 30 days after the nematode inoculation were similarly examined by using the second instar larvae of Scutellaria baicalensis (Example 1) and (Example 2). It was According to this, there was no difference in the number of four species of nematodes grown on the 15th day of inoculation, but on the 30th day, the number of the nematodes OJ-N2 was 480, which was about 2 to 10 times that of the conventional nematodes.
It proliferated in large numbers with ten thousand.

The results are shown in Table-3.

[Effects of the Invention] As described above, the insect parasitic nematode Hetero-rhabditis spp. (OJ-N) detected in soil under natural conditions in Japan.
2) has a high insecticidal activity against various insects, especially the nematode difficult Coleoptera pests, and long-term viability under natural conditions, and since it can be mass-cultured, it is a biological control agent. It is extremely effective to use it as a means.

Furthermore, the inventors of the present invention previously disclosed in JP-A-60-260678.
As shown in No. 6, it has been found that an excellent effect can be obtained by using an insect-parasitic nematode as a soil improving agent loaded on park compost. However, this nematode OJ-N2 also produces bark compost and the like. It goes without saying that the effect is improved by using it together with the fermented compost of.

[Brief description of drawings]

FIG. 1 is an enlarged photograph of an infectious larva of the insect parasitic nematode OJ-N2 used in the present invention, FIG. 2 is a first-generation female adult OJ-N2, and FIG. An enlarged photograph, Fig. 4,
Second generation adult OJ-N2, FIG. 5 shows the same tail (B)
Is an enlarged photograph of.

Claims (1)

[Claims] 1. An insect parasitic nematode, Heterorhabditis spp. OJ.N.
A method for biologically controlling insects using 2.