KR20070115489A - Novel strain of cordyceps sp. and method for preventing plant strongylosis using the same - Google Patents

Abstract

A novel strain of Cordyceps sp. is provided to control plant strongylosis caused by longicorn beetle insects in an environment-friendly manner by exterminating larva, pupa and adult insect of longicorn beetle insects within a short period of time. A novel strain of Cordyceps militaris Hope(KCTC 18113P) capable of controlling plant strongylosis is isolated from the longicorn beetle insects by sterilizing the surface of longicorn beetle insects with ethylalcohol, sterilizing the longicorn beetle insects with pure ethylalcohol and a flame under antiseptic condition, and extracting body fluid from the longicorn beetle insects through injection. The plant strongylosis is controlled by inoculating Cordyceps militaris Hope(KCTC 18113P) to needle-leaf trees for 5-30 days.

Description

Bacillus spp. Strain controlling the sky insects and a method for controlling plant nematode disease using the same {Novel strain of Cordyceps sp. and method for preventing plant strongylosis 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 is a new genus Cordyceps for controlling sky insects. sp . The present invention relates to a strain and a method for controlling plant nematode disease using the same, and more particularly, to a new strain of Bacillus sp. Isolated from the skyworm insects, spores and carcass extracts of the bacterium, their preparation method, and these as pesticides for controlling nematode diseases. The present invention also relates to a method for controlling plant nematode disease using spores and carcass extracts of Bacillus.

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.

Accordingly, the present inventors have made efforts to develop environmentally friendly pesticides, and as a result, they have been isolated from the insects of celestial insects infected with the plant reptile disease ( Cordyceps). sp . ) Isolate the strains and incubate them in artificial medium to collect Leprotococcus spores, and then inoculate them into larvae, pupa and adults of the sky bird, and observe that all of them are controlled with high mortality within a short period of time. The present invention was successfully completed by confirming that pine re-nematode control agent containing as an active ingredient is useful as a biochemical pesticide.

The present invention is a novel genus Cordyceps isolated from the sky insect sp . ) Strains, spores and carcass extracts of the Bacillus bacterium, their preparation method, the use of them as a pesticide for the control of plant reptile disease, and to provide a method for controlling plant nematode disease using the spores and carcass extract of the Leukemia bacteria.

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 is a strain of the genus Bacillus ( Cordyceps sp . ) Is used to control plant worm nematodes. At this time, the re-nematode disease includes pine re-nematode disease and pine re- nematode disease.

The present invention is the strain of the genus Bacillus (Cordyceps sp .Provided is a nematode disease control agent comprising, as an active ingredient.

Specifically, the present invention provides a natural nematode or artificially cultured spores of the Bacillus sp. Strain as a active nematode disease control agent.

The present invention also provides a re-nematode disease control agent comprising as an active ingredient a body fluid and / or an extract collected from an infected skyworm insect.

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 addition, the present invention is the genus Cordyceps sp . ) Provides a control method for controlling re-nematode disease using a strain. 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 provides a control method for controlling re nematode disease using the body fluids and / or cadaveric extracts collected from natural spores or artificially cultured spores of Leukemia bacteria and celestial insects infected with Reptile nematode disease as the S. aureus strains. do.

The present invention is inoculated with the strains of the genus Bacillus provides a control method of nematode disease is preferably continued for 5 to 30 days.

The control method of the present invention can be used for all chipping forests and the like to cause re-nematode disease, 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 addition, the present invention is the genus Cordyceps sp . ) Provides a biochemical pesticide that controls re-nematode disease using the 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. Pine Nematode disease  Preparation of Pesticide Composition for Control 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. Pine Nematode disease  Preparation of Pesticide Composition for Control 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. Pine Nematode disease  Preparation of pesticide composition for control 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 provides a new genus Cordyceps isolated from the sky insects. sp . ) Strains, spores and carcass extracts of the Bacillus Bacillus, their preparation method, the use of them as a pesticide for the control of nematode disease, and the method of controlling plant nematode disease using the spores and cabbage extract of Bacillus Bacillus, In this method, the larvae, chrysalis and adult insects of skyworm insects, which mediate plant reptile diseases, can be effectively killed with a high mortality rate in a short period of time. In addition to being able to produce at low cost, it is possible to eliminate the chemical control method using existing mef and sprite without damaging the livestock and to solve the problem of ecosystem destruction by overuse of chemical pesticide. As it has an effect, it is industrially very useful.

Claims (14)

Cordyceps with Control Effect on Plant Reptile Nematode Disease sp . ) Strains. The method of claim 1 wherein the strain is in baekganggyun cor di sepseu Ms other less (Cordyceps militaris) is the strain, preferably cor-di sepseu Militaris Hope ( Cordyceps militaris Hope) strain of Bacillus genus, characterized in that the strain. The method of claim 1 or claim 2, wherein the strain is in baekganggyun cor di sepseu Militaris Hope ( Cordyceps militaris Hope) # 1 strain of the genus Bacillus, characterized in that (accession number: KCTC 18113P). Cordyceps of claim 1 sp . ) Plant re nematode disease control agent comprising the strain as an active ingredient. The method of claim 4, wherein the strain of the genus Bacillus sp. Natural spores and artificial cultured spores, characterized in that the plant nematode disease control agent. 5. The plant repellent nematode disease control agent according to claim 4, wherein the strain of the genus Leprosy bacterium comprises body fluids and / or extracts collected from the skyworm insects infected with the nematode disease. 7. The plant reptile nematode control agent according to claim 6, wherein the sky insect includes larvae, pupa and / or adults. A corpse extract used to control plant worm nematodes, obtained by extracting juice from dead skyworm insects infected with plant worms. (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) stabbing a sterile syringe needle into the insect torso; Process for obtaining the body extract of claim 8 by the process. Cordyceps strain of claim 1 sp . Control method for controlling plant worm nematode disease. The method of claim 10, wherein the strain of the genus Bacillus comprises natural spores and artificially cultured spores. 11. The method of claim 10, wherein the strain of the genus Leprosy bacterium comprises a body fluid and / or a corpse extract taken from an arachnid insect infected with a plant worm nematode disease. The method of claim 10, wherein the strain of the genus Bacillus is inoculated and treated for a period of 5 days to 30 days. Cordyceps strain of claim 1 sp . Biochemical pesticides controlling plant reptile diseases with) as an active ingredient.

Images