KR100854721B1 - Novel strain of Cordyceps sp. and method of killing a long-horned beetle using the same - Google Patents

Abstract

The present invention relates to a new Cordyceps sp. Strain that controls anthropogenic insects and a method of insecticidal insects of anthropogenic insects causing a plant nematode disease using the same, and more particularly, a new Bacillus isolated from anthropogenic insects. The present invention relates to a genus strain, spores and carcass extracts of the Bacillus bacterium, their preparation methods, the use of them as pesticides for the control of nematode disease, and a pesticidal method of skyworm insects that mediates the plant nematode disease using the spores and carcass extracts of Bacillus sp. . The control method using Bacillus sp. Strains of the present invention can control all the larvae, pupa and adults of the sky insects that mediate plant worm nematode disease with high mortality in a short period of time at a low cost and extracts the Bacillus, its spores and carcasses The back light is developed as an environmentally friendly biological pesticide that can be widely used to eradicate pine nematode disease.

Cordyceps sp. Strains, spore and carcass extracts of Leukemia bacteria, pine worm nematode disease, antler insects, methods of controlling plant worm nematodes, biological pesticides

Description

Bacillus spp. Strain controlling the sky insects and insecticide method of the sky insects using the same {Novel strain of Cordyceps sp. and method of killing a long-horned beetle using the same}

1 is a genus of Bacillus Bacillus of the present invention ( Cordyceps sp . ) Shows the process (A and B) of infecting larvae of larvae using spores and carcass extracts of the strain, and the larvae carbonized by the infection (C).

Figure 2 is a genus of Bacillus Bacillus of the present invention ( Cordyceps sp . ) Shows the process of inoculating the larvae of the sky larvae and 0, 15, 30 and 45 days after the larvae are killed and carbonized to form a mycelium.

The present invention relates to a new Cordyceps sp. Strain that controls anthropogenic insects and a method of insecticidal insects of anthropogenic insects causing a plant nematode disease using the same, and more particularly, a new Bacillus isolated from anthropogenic insects. The present invention relates to a genus strain, spores and carcass extracts of Bacillus sp., The preparation method thereof, their use as pesticides for the control of nematode disease, and methods of insecticidal insects of celestial insects that cause plant re nematode disease using spores and carcass extracts of Bacillus sp. .

Pine nematode disease is a plant disease in which red nematodes invade pine trees by invading pines, destroying resinous spheres and ducts in wood, feeding on intracellular fluids, preventing sap and water from moving, and turning the leaves into yellowish brown and resin flowing. The pine re-nematode disease is also called pine AIDS or pine foot and mouth disease, and once infected trees are damaged by physical and chemical damages such as completely dried wood parts that lose moisture and lose weight, and blue wood deformation inside the wood. It is a fatal disease that must be killed within a year.

Pine nematodes are small, about 1 mm long, have a thread-like shape, and are characterized by the rapid growth of two animals into 200,000 animals at 25 ° C. in 20 days without being conspicuous.

Since the pine re-nematode was first introduced in Japan in 1905, most pine trees endangered, causing severe damage to various pines. In fact, Korea landed on Geumjeongsan in Busan in 1988 and spread to nearby Si-gun, causing serious damage. In addition, this pine AIDS has caused severe damage to pine forests near Yudalsan, Mokpo, Jeonnam, including Busan and Gyeongnam. For reference, in Japan, about 2.4 million cubic meters of pine and algae have been killed annually since 1941, and in China, after the first occurrence in Nanjing in 1982, about 7 million hectares of horsetail and sea pine forest Continue to cause severe damage. Also in Taiwan, a pine re-nematode disease occurred in 1985, endangering the special species, Yugu Song.

In Korea, pine re-nematodes were designated as prohibited pests under the Plant Protection Act and tried to prevent their invasion. Nevertheless, the disease has a tremendous ability to infect and spread, and therefore, there are many difficulties in preventing the influx and it is still failing today. However, if left untreated, plant nematode disease spreads throughout the southern region in the next few years, contaminating coniferous forests, including pines and pines, which account for about 42% of the forest area, causing significant loss of forest resources and destruction of the natural environment. It is expected to lead to economic and social poverty.

Moreover, in China and Japan, pine re-nematodes have been defined as plant diseases for about 40 years, and various studies have been conducted on their development process, life history, and control methods, but in Korea, professional research is still very early. Therefore, more intensive investment and research are urgently needed.

Therefore, the present inventors continued to develop eco-friendly pesticides, and as a result, isolates of the strains of Cordyceps sp. Inoculates larvae, chrysalis and adult insects and observes that all of them are controlled with high mortality within a short period of time and control pine re-nematodes by using insecticides of celestial insects whose active ingredients are Leukococcus, its spores and carcass extracts. The invention was successfully completed by confirming that it is useful.

The present invention is a new Cordyceps sp. Strain isolated from the sky insects, spores and carcass extracts of the Bacillus, their production method, the use of them as pesticides for controlling plant nematode diseases by insecticides And it is an object of the present invention to provide an insecticidal method of sky bird insects using the spores and carcass extract of the Bacillus.

In order to achieve the above object, the present invention is a genus Cordyceps also called Cordyceps genus having insecticidal effect on sky insects ( Cordyceps sp . ) Provides the strain.

Preferably, the present invention refers, also known as cor di sepseu "Cordyceps militaris" Millie other leases (Cordyceps militaris) provide a strain, and more preferably cor-di sepseu ms other less hops (Cordyceps militaris Hope) strain.

The present invention as a pesticidal effect on the sky insects, the genus Cordyceps excellent in the control effect against the plant nematode disease ( Cordyceps sp . ) Provides the strain.

The skyworm insects include medium insects skyworms and brushed skyworm insects, and the like, and preferably include sky cows, stink bugs and bees, and in addition to all insects that may cause plant reptile diseases, including pine re-nematode diseases. Can be.

The present invention is Cordyceps Millie other leases Hope (Cordyceps militaris Hope) strain was named Chrysalis of the genus Cordyceps Cordyceps sinensis # 1 and was deposited with the Korea Biotechnology Research Institute Genetic Bank on May 22, 2006 (Accession No .: KCTC 18113P).

In addition, the present invention provides a use of the Leukemia strain ( Cordyceps sp. ) To insects of the skyworm insects that mediate plant reptile disease. At this time, the re-nematode disease includes pine re-nematode disease and pine re- nematode disease.

The present invention provides a pesticide of the sky bird insects having the above-mentioned Leukemia strain ( Cordyceps sp. ) As an active ingredient.

Specifically, the present invention provides a natural insecticidal or spores of artificial cultured spores of the genus Bacillus strain as an active ingredient.

The present invention also provides an insecticide of the skyworm insects, which contains the body fluids and / or extracts from the infected skyworm insects as an active ingredient.

The skybird insects include antler skyworms and beetle skybird insects, and in particular, skybirds, stink bugs and bees, and the like, and their larvae, pupa and / or adults, respectively.

In addition, the present invention is the genus Cordyceps sp . ) Provides a separation method for obtaining a strain.

Specifically, the present invention

(1) torso of tortoiseshell insects;

(2) sterilizing the surface of the insect with ethyl alcohol;

(3) sterilizing again with pure ethyl alcohol, flame or the like in aseptic conditions;

(4) making a small incision in the tortoiseshell insect; And

(5) incubated in liquid medium; It provides a manufacturing method for separating the white bacterium by the process.

In the process (5), it is preferable to use a liquid PDA medium as the liquid medium, the temperature is preferably incubated in the range of 20 to 30 ℃, it is more preferable to incubate at 25 ℃, the period is 5 to 10 days It is preferred to continue incubating for a while.

In addition, the present invention is the genus Cordyceps sp . It provides a method for obtaining natural spores of artificial strain and artificially cultured spores.

Specifically, the present invention

(1) separating leukemia by the above procedure;

(2) mixing the sky larvae with the liquid medium;

(3) inoculating the Bacillus bacterium using a sterilized container; And

(4) sterile cultured; The process provides a method of separating the spores of Bacillus Bacillus.

In the process (2), it is preferable to use a liquid PDA medium as the liquid medium, the temperature is preferably cultured in the range of 20 to 30 ℃, more preferably incubated at 25 ℃, the duration is 2 to 4 months It is preferred to continue incubating for a while.

The present invention also provides a carcass extract used to control the plant worm nematode disease obtained by extracting the juice from sky insects infected with worm nematode disease.

In addition, the present invention provides a method for obtaining carcass extract by extracting the juice from dead sky insects infected with re-nematode disease.

Specifically, the present invention

(1) obtaining carcasses of the skyworm insects;

(2) sterilizing the surface of the insect with ethyl alcohol;

(3) sterilization with pure ethyl alcohol, flame or the like in aseptic conditions; And

(4) stabbing a sterile syringe needle into the trunk of the insect carcass; It provides a process for obtaining the body fluid and / or corpse extract of insects containing Leukemia as a process.

The skybird insects include antler skyworms and beetle skybird insects, and in particular, skybirds, stink bugs and bees, and the like, and their larvae, pupa and / or adults, respectively.

In another aspect, the present invention provides a method of insecticidal insects of the sky birds using the Cordyceps sp . At this time, the re-nematode disease includes plant re-nematode disease, preferably pine re-nematode disease and pine re- nematode disease.

Specifically, the present invention uses the natural spores or artificially cultured spores of the Bacillus sp. Mycelium strains, and body fluids and / or cadaveric extracts collected from the skyworm insects infected with the nematode disease. Provide a way to kill insects.

The present invention inoculates the strains of the genus Bacillus provides an insecticidal method of the skyworm insects that mediate the re-nematode disease, which is preferably continued for 5 to 30 days.

The insecticidal method of the present invention can be used for all chipping forests, including pine and pine. The strain of Bacillus sp. Inoculated at this time may be appropriately adjusted according to the plant or sky insect.

Depending on the control method of the present invention in the baekganggyun (Cordyceps sp . ) When treated with the strain, its spores and / or carcass extracts, the sky larvae are browned in the head and body in the early stages, drain the body fluids in the middle stages, and later become brownish, dying in the process of lustrousness and motility in the process. Has a slowing feature. In addition, the inoculated strain of the genus Leukemia, its spores and / or carcass extract, and the like is carbonized in black within 30 days and thus carbonized to form a white mycelium within 45 days of death.

According to embodiments of the present invention, the control method has a mortality rate of about 78% ± 10%, preferably within 5 to 15 days, more preferably within about 10 days.

In another aspect, the present invention provides a biochemical pesticide for insecticides of the skyworm insects to mediate the re-nematode disease by using the Cordyceps sp. Strain as an active ingredient. At this time, the re-nematode disease includes plant re-nematode disease, preferably pine re-nematode disease and pine re- nematode disease.

The biochemical pesticides of the present invention can be applied to all plants causing the nematode insects to mediate re nematode disease. In addition, the biochemical pesticides can be used for insecticidal and disinfection of various insects including the skyworm insects.

The biochemical pesticide preparation of the present invention is mixed with 5 to 90% by weight of the dry powder or culture medium of the strains of Bacillus sp., Spores and / or carcasses thereof, according to a conventional method, a surfactant, an inorganic salt, an auxiliary agent, a binder, an extender, and the like are mixed. It can be provided as a microbial composition prepared by. In addition, the microbial composition comprising the dry powder or culture of the strain of the present invention can be prepared in various formulation forms, that is, liquids, hydrates, powders, granules and emulsions by adjusting the respective additives.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example  1. Isolation and Culture of Bacillus Strains

The present invention baekganggyun (Cordyceps causing disease Bursaphelenchus xylophilus militaris ) strains were obtained, the trunks of the infected sea salted blue fish were first transferred to the inoculation room by surface sterilization using 70% ethyl alcohol. It was then placed in a sterile petri dish previously sterilized, and then again sterilized again with pure ethyl alcohol, and then sterilized again with a flame, and then the trunk of the brushfish skyfish was cut into a 3 mm length with a scalpel. . Thus cut pieces of insects were put in a liquid PDA medium prepared in advance one by one and incubated for one week at 25 ℃ and subcultured again using artificial medium.

Baekganggyun strains isolated by the above process is cor di sepseu Millie other leases Hope (Cordyceps militaris Hope) was identified as a strain and named pupa cordyceps sinensis # 1 was deposited on May 22, 2006, gene bank affiliated with the Korea Biotechnology Research Institute (Accession Number: KCTC 18113P).

Example  2. Collection of Bacillus spores

The present invention is added to the liquid PDA medium and sky larva larvae at a ratio of 10: 5 (V / V) of the isolated and artificially cultured Baeksiyun bacteria strains in Example 1 to obtain spores of Bacillus retinopathy strains causing pine re nematode disease The prepared mixed medium was inoculated into a sterilized culture flask, which was put therein, and then aseptically cultured at 25 ° C. for 3 months to collect leukococcus spores.

Example  3. Collection of Carcass Extracts Containing Bacillus Strains

The present invention provides larvae, adults and chrysalis of the skyworm insects with pine worm nematode disease, larvae, adults and chrysalis of algae insects already dead due to pine worm nematode disease The treatment was carried out as follows to obtain a dark brown body fluid or extract therefrom. In addition, the larvae, adults and pupae of the skyworm insects were directly inoculated with the Bacillus spores obtained in Example 2 to cause the disease, thereby killing dead insect carcasses. In this process, the same procedure as in Example 1 was applied in order to minimize the incorporation of other strains, the surface of dead insects were sterilized, a needle was inserted into the trunk, and a dark brown body fluid or dead body extract containing the Leukemia strain was collected.

Example  4. Investigation of Mortality of Skyborne Insects Using Leukococcal Strains

The present invention, in order to investigate the mortality rate of the sky larvae using the spores or carcass extract of the Bacillus Bacillus strain, each of the three groups of small chalet larvae were inoculated by inoculating the Bacillus sp. Breeding for 45 days at room temperature of about 20 ℃. In order to investigate the mortality of the larvae, dark brown juice solution from the carcasses was collected after the larvae died, diluted 10-fold and inoculated into healthy larvae and examined for mortality.

As a result of artificial inoculation of skyworm larvae, the natural insect bacterium, including the sky cow, stink bug and bee, was found to be about 78%. In addition, as a result of artificial inoculation of the extracts from the carcasses of the skyworm insects to the skyworm larvae, the insecticidal rate of the larvae was measured to be about 94%, and the significance between treatment groups was high. It was confirmed that there is no big difference.

Example  5. Investigation of lethal process of skyworm insects

In order to investigate the lethal process of skyworm larvae using spores or carcass extracts of the Bacillus sp. Strain of the present invention, 6 skyworm larvae were put into each repetition group, and mycelia of Bacillus Bacillus cultured at 25 ° C. for 45 days using silkworm pupa. Was inoculated by diluting 10-fold. Then, the infected larvae were reared in a breeding box at about 20 ° C. for 45 days, and the larvae were infected with the Leukemia strains, and the first expression time, damage pattern, damage symptoms, and lethal process were observed in detail.

As a result, the inoculation of Bacillus Bacillus strains of the present invention, artificial breeding in a breeding box of about 20 ° C., and then the first damage manifestation (sign) appearing in the sky larvae was 7 days after the inoculation, and then the larva was slowed in motility. As a result, it was gradually died and the death time was 9 days. The mortality rate was about 78% when the total 15 days after the infection occurred.

As a specific type of damage, the head and torso of the larvae were partially browned at the beginning of the infection, and the body fluid was leaked afterwards. The carcasses of the larvae were carbonized to give black and white mycelium.

Example 6 Preparation of Pesticide Composition for Insecticide of Skyworm Insects for Controlling Pine Reptile Disease I

In the present invention, Bacillus sp. Strain (Accession No .: KCTC 18113P), 5 to 60% by weight of the culture or dry powder of its spores or dead bodies extract, 0 to 40% by weight of inorganic salts, 5 to 30% by weight of surfactant, the remaining amount The extenders were mixed uniformly and milled to produce a biochemical pesticide composition in the form of a hydrate.

Example 7 Preparation of Pesticide Composition for Insecticide of Skyworm Insects for Controlling Pine Reptile Disease II

In the present invention, Bacillus sp. Strain (Accession No .: KCTC 18113P), 5 to 60% by weight of the culture solution or dry powder of spores or carcass extract thereof, 0 to 40% by weight of inorganic salts, 5 to 30% by weight of surfactant, 1 to The biochemical pesticide composition in the form of granular hydrated powder was prepared by uniformly mixing, grinding, kneading, and kneading the extender with 10% by weight of binder and remaining amount.

Example 8 Preparation of Pesticide Composition for Insecticides of Sky Bird Insects for Control of Pine Needle Nematode Disease III

In the present invention, Bacillus sp. Strain (Accession number: KCTC 18113P), 5 to 50% by weight of the culture solution or dry powder of spores or dead bodies extract thereof, 0 to 30% by weight of inorganic salts, 5 to 30% by weight of surfactant, 1 to 70% by weight of a pulverized portion obtained by wet grinding a mixture of 10% by weight of an extender and a residual amount of water, and 1 to 20% by weight of a binder, 1 to 20% by weight of an antifreeze agent and a balance of water. A biochemical pesticide composition was prepared in the form of a liquid hydrating agent which was uniformly mixed with 30% by weight of the thickened portion.

As described above, the present invention is a new Cordyceps sp. Strain isolated from the sky insects, spores and carcass extracts of the Bacillus bacterium, their production method, these as pesticides for the insects of the sky insects that mediate the nematode disease The present invention relates to a method of insecticidal insects that mediate plant reptile disease using spores and carcass extracts of Bacillus sp. , Pupa and adult can be effectively exterminated in a short period of time with high mortality. The Bacillus strain, its spores and carcass extracts can be produced at low cost as an environmentally friendly biological pesticide, and do not harm the livestock. Chemicals using meth and sprites It is very useful industrially because it has the effect of eliminating the control method and further solving the problem of ecosystem destruction by overuse of chemical pesticides.

Claims (14)

delete delete Cor di sepseu ms other less hops (Cordyceps militaris Hope) # 1 strain (accession number: KCTC 18113P). The insecticidal composition of the sky bird insect which mediates a plant worm nematode disease, comprising the strain of claim 3 or its spores as an active ingredient. The insecticidal composition according to claim 4, wherein the spores are natural spores and artificially cultured spores. An insecticidal composition of a skyworm insect that mediates a plant worm nematode disease, comprising as an active ingredient a bodily fluid obtained from a skyworm insect infected with a plant worm nematode disease, a corpse extract of the skyworm insect, and a combination thereof. The insecticidal composition according to claim 4 or 6, wherein the sky insect is a larva, pupa, an adult or a combination thereof. Insecticidal carcass extracts of the skyworm insects obtained by extracting the juice from the skyworm insects that have been infected with the plant worm nematode disease. (1) obtaining carcasses of the skyworm insects; (2) sterilizing the surface of the insect with ethyl alcohol; (3) sterilization again with pure ethyl alcohol and flame in aseptic condition; And (4) puncturing the syringe needle sterilized by the insect torso; A method for preparing the carcass extract of claim 8. A method for insecticidal insects that mediates plant reptile disease using the strain of claim 3 or its spores. The method of claim 10, wherein the spores are natural spores and artificially cultured spores. The method of claim 10 or 11, wherein the strain or its spores are treated by inoculation for a period of 5 to 30 days. A method of killing skyworm insects that mediate plant worm nematode disease using body fluids, carcass extracts, or combinations thereof obtained from dead skyworm insects infected with plant worms. The method of claim 13, wherein the body fluid, carcass extract, or a combination thereof is inoculated for a period of 5 days to 30 days for treatment.

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